Human MEKK proteins, corresponding nucleic acid molecules, and uses therefor

ABSTRACT

Isolated nucleic acid molecules encoding human MEKK proteins, and isolated MEKK proteins, are provided. The invention further provides antisense nucleic acid molecules, recombinant expression vectors containing a nucleic acid molecule of the invention, host cells into which the expression vectors have been introduced and nonhuman transgenic animals carrying a human MEKK transgene. The invention further provides human MEKK fusion proteins and anti-human MEKK antibodies. Methods of using the human MEKK proteins and nucleic acid molecules of the invention are also disclosed, including methods for detecting human MEKK activity in a biological sample, methods of modulating human MEKK activity in a cell, and methods for identifying agents that modulate the activity of human MEKK.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of prior-filed provisionalapplication U.S. patent application Ser. No. 60/078,153 entitled “HumanMEKK2 Nucleic Acid and Protein Molecules and Uses Therefor”, filed Mar.16, 1998 and of prior-filed provisional application U.S. patentapplication Ser. No. 60/099,165 entitled “Human MEKK3 Protein andNucleic Acid Molecules and Uses Therefor” filed Sep. 4, 1998. Thepresent application is also related to PCT patent application Ser. No.PCT/US99/02974, entitled “MEKK1 Proteins and Fragments Thereof for Usein Regulating Apoptosis”, filed Feb. 12, 1999, which claims priority toU.S. application Ser. No. 09/023,130 entitled “Method And Product ForRegulating Apoptosis”, filed Feb. 13, 1998. The contents of theabove-referenced patent applications are incorporated herein by thisreference in its entirety.

FIELD OF THE INVENTION

This invention relates to isolated nucleic acid molecules encoding MEKKproteins, substantially pure MEKK proteins, and products and methods forregulating signal transduction in a cell.

BACKGROUND OF THE INVENTION

Mitogen-activated protein kinase (MAPKs) (also called extracellularsignal-regulated kinases or ERKs) are rapidly activated in response toligand binding by both growth factor receptors that are tyrosine kinases(such as the epidermal growth factor (EGF) receptor) and receptors thatare coupled to heterotrimeric guanine nucleotide binding proteins (Gproteins) such as the thrombin receptor. In addition, receptors like theT cell receptor (TCR) and B cell receptor (BCR) are non-covalentlyassociated with src family tyrosine kinases which activate MAPKpathways. Specific cytokines such as tumor necrosis factor (TNFα) canalso regulate MAPK pathways. The MAPKs appear to integrate multipleintracellular signals transmitted by various second messengers. MAPKsphosphorylate and regulate the activity of enzymes and transcriptionfactors including the EGF receptor, Rsk 90, phospholipase A₂, c-Myc,c-Jun and Elk-1/TCF. Although the rapid activation of MAPKs by receptorsthat are tyrosine kinases is dependent on Ras, G protein-mediatedactivation of MAPK appears to occur through pathways dependent andindependent of Ras.

The MAPKs are activated by phosphorylation on both a threonine andtyrosine by dual specificity kinases, MAPK/ERK kinases (MEKs) which are,in turn, activated by serine/threonine phosphorylation MAPK kinasekinases (MKKKs or MEKKs). At present, at least four MEKKs have beenidentified. The four MEKK proteins range from 69.5-185 kDa in size,having their kinase domains in the carboxy-terminal end of the proteinand their catalytic domains in the amino-terminal end of the protein.Murine MEKK1 was cloned initially on the basis of its homology with theSTE11 and Byr2 kinases from yeast (Lange-Carter et al. (1993) Science260:315-319; Xu et al. (1996) Proc. Natl. Acad. Sci. USA 93:5291-5295;and Blank et al. (1996) J. Biol. Chem. 271:5361-5368). Murine MEKK2 andMEKK3 were subsequently cloned and found to have 94% homology in theirkinase domains as well as 65% homology within their catalytic domains.Blank et al., supra. The cloning of murine MEKK4 revealed approximately55% homology to the kinase domains of MEKKs 1, 2, and 3 whereas theamino-terminal region of MEKK4 has little sequence homology to the otherMEKK family members. Gerwin et al. (1997) J. Biol. Chem. 272:8288-8295.MEKK1 and MEKK4, but not MEKK2 and MEKK3, bind to the low molecularweight GTP-binding proteins Cdc42 and Rac. Furthermore, MEKK1 also bindsto Ras in a GTP-dependent manner (Russell et al. (1996) J. Biol. Chem.11757-11760) and Ras activity is required for EGF-mediated stimulationof MEKK1 activity (Lange-Carter and Johnson (1994) Science265:1458-1461). In addition to growth factor receptor tyrosine kinases(i.e. EGF receptor), the TNF receptor, the FcεR1 in mast cells Ishizukaet al. et al. (1996) J. Biol. Chem. 271:12762-12766) and the N-formylmethionyl leucine peptide receptor in neutrophils have been shown toactivate MEKK1. EGF and TNF also activate MEKK3 and it also appears thatthe other MEKK proteins are regulated by tyrosine kinase receptors butthe intermediate components and effector molecules leading to theiractivation are poorly understood.

The cellular effects of MEKK1 are quite diverse and can be classified asbeing either JNK-dependent or JNK-independent. For example, MEKK1 canmediate activation of ERKI and ERK2 and, by a yet undefined mechanism,activation of the c-Myc transcription factor independent of JNK activity(Lassignal-Johnson et al. (1996) J. Biol. Chem. 271:3229-3237 andLange-Carter et al. (1993) Science 260:315-319). Alternatively, MEKK1may or may not require JNK activity for activation of IKB kinase whichleads to NKκB activation (Liu et al. (1996) Cell 87:565-576 and Meyer etal. (1996) J. Biol. Chem. 271 :8971-8976). Furthermore, depending uponthe cell type, MEKK1, but not MEKK2, 3 or 4, has been shown to mediateapoptosis by both JNK-dependent and JNK-independent mechanisms (Xia etal. (1995) Science 270:1326-1331 and Lassignal-Johnson et al. (1996) J.Biol. Chem. 271:3229-3237).

Given the important role of members of the MAPK signal transductioncascade, in particular the MEKK signal transduction molecules, inregulating mammalian cellular processes ranging from cellularproliferation and differation to cellular apoptosis, there exists a needfor identifying human MEKK nucleic acid and protein molecules as well asfor modulators of such molecules for use in regulating a variety ofhuman cellular responses.

SUMMARY OF THE INVENTION

This invention provides human MEKK compositions. In particular, thisinvention provides isolated nucleic acid molecules encoding human MEKK1,human MEKK2, and human MEKK3. The invention further provides isolatedhuman MEKK1, human MEKK2, and human MEKK3 proteins. Because the MEKKcompositions of the invention are human-derived, they function optimallyin human cells (compared with non-human MEKK compositions) and do notstimulate an immune response in humans.

One aspect of the invention pertains to an isolated nucleic acidmolecule having a nucleotide sequence which encodes a human MEKKprotein. In a preferred embodiment, the nucleic acid molecule has thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. Inother embodiments, the nucleic acid molecule has at least 90-91%nucleotide identity, more preferably 92-93% nucleotide identity, morepreferably 94-95% nucleotide identity, more preferably 96-97% nucleotideidentity, more preferably 98-99% nucleotide identity, and even morepreferably 99.5% nucleotide identity with the nucleotide sequence SEQ IDNO:1, SEQ ID NO:3, or SEQ ID NO:5.

The isolated nucleic acid molecules of the invention encoding human MEKKproteins can be incorporated into a vector, such as an expressionvector, and this vector can be introduced into a host cell. Theinvention also provides a method for producing a human MEKK protein byculturing a host cell of the invention (carrying a huMEKK1, huMEKK2, orhuMEKK3 expression vector) in a suitable medium until a human MEKKprotein is produced. The method can further involve isolating the humanMEKK protein from the medium or the host cell.

Another aspect of the invention pertains to an isolated human MEKKproteins. Preferably, the human MEKK protein has the amino acid sequenceof SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6. In other embodiments, theprotein has at least 90-91% amino acid identity, more preferably 92-93%amino identity, more preferably 94-95% amino identity, more preferably96-97% amino identity, more preferably 98-99% amino identity, and evenmore preferably 99.5% amino acid identity with the amino acid sequenceof SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

Fusion proteins, including a human MEKK protein operatively linked to apolypeptide other than human MEKK, are also encompassed by theinvention, as well as antibodies that specifically bind a human MEKKprotein. The antibodies can be, for example, polyclonal antibodies ormonoclonal antibodies. In one embodiment, the antibodies are coupled toa detectable substance.

Another aspect of the invention pertains to a nonhuman transgenic animalthat contains cells carrying a transgene encoding a human MEKK protein.

Yet another aspect of the invention pertains to a method for detectingthe presence of human MEKK in a biological sample. The method involvescontacting the biological sample with an agent capable of detecting anindicator of human MEKK activity such that the presence of human MEKK isdetected in the biological sample. The invention also provides a methodfor modulating human MEKK activity in a cell which involves contactingthe cell with an agent that modulates human MEKK activity such thathuman MEKK activity in the cell is modulated.

Still another aspect of the invention pertains to methods foridentifying a compound that modulates the activity of a human MEKKprotein. These methods generally involve: providing an indicatorcomposition that comprises a human MEKK protein; contacting theindicator composition with a test compound; and determining the effectof the test compound on the activity of the human MEKK protein in theindicator composition to thereby identify a compound that modulates theactivity of a human MEKK protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B depicts the cDNA sequence of human MEKK1. The nucleic acidsequence corresponds to nucleotides 1 to 3911 of SEQ ID NO:1.

FIG. 2 depicts the amino acid sequence of human MEKK1. The amino acidsequence corresponds to amino acids 1 to 1302 of SEQ ID NO:2.

FIGS. 3A-3J show a global alignment of the nucleic acid sequence ofhuman MEKK (SEQ ID NO:1) with the nucleic acid sequence of mouse MEKK1SEQ ID NO:7).

FIGS. 4A-4C show an alignment of the amino acid sequence of human MEKK1(SEQ ID NO:2) with that of murine MEKK1 (SEQ ID NO:8). Amino aciddifferences between the two sequences are underlined and bolded.

FIG. 5 depicts the cDNA sequence of human MEKK2. The nucleic acidsequence corresponds to nucleotides 1 to 2013 of SEQ ID NO:3.

FIG. 6 depicts the amino acid sequence of human MEKK2. The amino acidsequence corresponds to amino acids 1 to 619 of SEQ ID NO:4.

FIGS. 7A-7E shows a global alignment of the nucleic acid sequences ofhuman MEKK2 (SEQ ID NO:3) and murine MEKK2 (SEQ ID NO:9).

FIG. 8 shows an alignment of the amino acid sequences of human MEKK2(SEQ ID NO:4) and murine MEKK2 (SEQ ID NO:10).

FIG. 9 depicts the cDNA sequence of human MEKK3. The nucleic acidsequence corresponds to nucleotides 1 to 1935 of SEQ ID NO:5.

FIG. 10 depicts the amino acid sequence of human MEKK3. The amino acidsequence corresponds to amino acids 1 to 626 of SEQ ID NO:6.

FIGS. 11A-11G show a global alignment of the nucleic acid sequences ofhuman MEKK3 (SEQ ID NO:5) and murine MEKK3 (SEQ ID NO:11).

FIG. 12 shows an alignment of the amino acid sequences of human MEKK3(SEQ ID NO:6) and murine MEKK3 (SEQ ID NO:12).

FIG. 13 shows an alignment of the amino acid sequences of the kinasecatalytic domains of murine MEKK1 (corresponding to amino acids1229-1493 of SEQ ID NO:8), murine MEKK2 (corresponding to amino acids361-619 of SEQ ID NO:10), murine MEKK3 (corresponding to amino acids367-626 of SEQ ID NO:12), murine MEKK4 (corresponding to amino acids1337-1597 of SEQ ID NO:13), human MEKK1 (corresponding to amino acids1038-1302 of SEQ ID NO:2), human MEKK2 (corresponding to amino acids361-619 of SEQ ID NO:4), and human MEKK 3 (corresponding to amino acids367-626 of SEQ ID NO:6). The consensus kinase domains are indicated bythe roman numerals I through XI. The most highly conserved residues areunderlined.

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to human MEKK compositions, such as isolatednucleic acid molecules encoding human MEKK1, human MEKK2, and humanMEKK3. The invention also pertains to isolated human MEKK proteins(e.g., human MEKK1, human MEKK2, and human MEKK3), as well as methods ofuse therefor. The human compositions of the invention have theadvantages that they function optimally in human cells (compared withnon-human MEKK compositions) and do not stimulate an immune response inhumans.

So that the invention may be more readily understood, certain terms arefirst defined.

As used herein, the term “human MEKK protein” is intended to encompassproteins that share the distinguishing structural and functionalfeatures (described further herein) of the human MEKK protein having theamino acid sequences of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6,including the amino acid residues unique to human MEKK proteins (ascompared to mouse MEKK proteins), which are underlined and bolded inFIG. 4, FIG. 8, and FIG. 12.

As used herein, the term “nucleic acid molecule” is intended to includeDNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g.,mRNA). The nucleic acid molecule may be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

An used herein, an “isolated nucleic acid molecule” refers to a nucleicacid molecule that is free of gene sequences which naturally flank thenucleic acid in the genomic DNA of the organism from which the nucleicacid is derived (i.e., genetic sequences that are located adjacent tothe gene for the isolated nucleic molecule in the genomic DNA of theorganism from which the nucleic acid is derived). For example, invarious embodiments, an isolated human MEKK nucleic acid moleculetypically contains less than about 10 kb of nucleotide sequences whichnaturally flank the nucleic acid molecule in genomic DNA of the cellfrom which the nucleic acid is derived, and more preferably containsless than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb ofnaturally flanking nucleotide sequences. An “isolated” human MEKKnucleic acid molecule may, however, be linked to other nucleotidesequences that do not normally flank the human MEKK sequences in genomicDNA (e.g., the human MEKK nucleotide sequences may be linked to vectorsequences). In certain preferred embodiments, an “isolated” nucleic acidmolecule, such as a cDNA molecule, also may be free of other cellularmaterial. However, it is not necessary for the human MEKK nucleic acidmolecule to be free of other cellular material to be considered“isolated” (e.g., a human MEKK DNA molecule separated from othermammalian DNA and inserted into a bacterial cell would still beconsidered to be “isolated”).

As used herein, the term “hybridizes under high stringency conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences having substantial homology to each otherremain stably hybridized to each other. Preferably, the conditions aresuch that sequences at least about 70%, more preferably at least about80%, even more preferably at least about 85% or 90% homologous to eachother typically remain hybridized to each other. A preferred,non-limiting example of high stringency conditions are hybridization ina hybridization buffer that contains 6×sodium chloride/sodium citrate(SSC) at a temperature of about 45° C. for several hours to overnight,followed by one or more washes in a washing buffer containing 0.2×SSC,0.1% SDS at a temperature of about 50-65° C.

To determine the percent homology of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence and non-homologous sequences can be disregardedfor comparison purposes). In a preferred embodiment, the length of areference sequence aligned for comparison purposes is at least 30%,preferably at least 40%, more preferably at least 50%, even morepreferably at least 60%, and even more preferably at least 70%, 80%, or90% of the entire length of the reference sequence (e.g., when aligninga second sequence to the MEKK amino acid sequence of SEQ ID NO:6 having626 amino acid residues, at least 188, preferably at least 250, morepreferably at least 313, even more preferably at least 376, and evenmore preferably at least 438, 501 or 563 amino acid residues arealigned). In a more preferred embodiment, the aligned amino acidresidues are consecutive (eg., homologous or identical over 188, 250,313, 376, 438, 501, or 563 consecutive amino acid residues.) Afteraligning, the amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are homologous at that position (i.e., asused herein amino acid or nucleic acid “homology” is equivalent to aminoacid or nucleic acid “identity”). The percent homology between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions×100).

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithim. A preferred, non-limiting example of a mathematicalalgorithim utilized for the comparison of sequences is the algorithm ofKarlin and Altschul (1990) Proc. Natl. Acad Sci. USA 87:2264-68,modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, word length=12 to obtain nucleotide sequenceshomologous to MEKK nucleic acid molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=50,word length=3 to obtain amino acid sequences homologous to MEKK proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

Another preferred, non-limiting example of a mathematical algorithimutilized for the comparison of sequences is the algorithm of Myers andMiller (1989) CABIOS. Such an algorithm is incorporated into the ALIGNprogram (version 2.0) which is part of the GCG sequence alignmentsoftware package. When utilizing the ALIGN program for comparing aminoacid sequences, a PAM120 weight residue table, a gap length penalty of12, and a gap penalty of 4 can be used. Another preferred, non-limitingexample of a mathematical algorithim utilized for the alignment ofprotein sequences is the Lipman-Pearson algorithm (Lipman and Pearson(1985) Science 227:1435-1441). When using the Lipman-Pearson algorithm,a PAM250 weight residue table, a gap length penalty of 12, a gap penaltyof 4, and a Ktuple of 2 can be used. A preferred, non-limiting exampleof a mathematical algorithim utilized for the alignment of nucleic acidsequences is the Wilbur-Lipman algorithm (Wilbur and Lipman (1983) Proc.Natl. Acad. Sci. USA 80:726-730). When using the Wilbur-Lipmanalgorithm, a window of 20, gap penalty of 3, Ktuple of 3 can be used.Both the Lipman-Pearson algorithm and the Wilbur-Lipman algorithm areincorporated, for example, into the MEGALIGN program (e.g., version3.1.7) which is part of the DNASTAR sequence analysis software package.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein).

As used herein, an “antisense” nucleic acid comprises a nucleotidesequence which is complementary to a “sense” nucleic acid encoding aprotein (e.g., complementary to the coding strand of a double-strandedcDNA molecule, complementary to an mRNA sequence or complementary to thecoding strand of a gene. Accordingly, an antisense nucleic acid canhydrogen bond to a sense nucleic acid).

As used herein, the term “coding region” refers to regions of anucleotide sequence comprising codons which are translated into aminoacid residues, whereas the term “noncoding region” refers to regions ofa nucleotide sequence that are not translated into amino acids (e.g., 5′and 3′ untranslated regions).

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively linked.Such vectors are referred to herein as “recombinant expression vectors”or simply “expression vectors”. In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” may be usedinterchangeably as the plasmid is the most commonly used form of vector.However, the invention is intended to include such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

As used herein, the term “host cell” is intended to refer to a cell intowhich a nucleic acid of the invention, such as a recombinant expressionvector of the invention, has been introduced. The terms “host cell” and“recombinant host cell” are used interchangeably herein. It should beunderstood that such terms refer not only to the particular subject cellbut to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to either mutationor environmental influences, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

As used herein, a “transgenic animal” refers to a non-human animal,preferably a mammal, more preferably a mouse, in which one or more ofthe cells of the animal includes a “transgene”. The term “transgene”refers to exogenous DNA which is integrated into the genome of a cellfrom which a transgenic animal develops and which remains in the genomeof the mature animal, for example directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal.

As used herein, a “homologous recombinant animal” refers to a type oftransgenic non-human animal, preferably a mammal, more preferably amouse, in which an endogenous gene has been altered by homologousrecombination between the endogenous gene and an exogenous DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal, prior to development of the animal.

As used herein, an “isolated protein” refers to a protein that issubstantially free of other proteins, cellular material and culturemedium when isolated from cells or produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized.

In one embodiment, a MEKK protein is identified based on the presence ofat least a “catalytic domain” in the protein or corresponding nucleicacid molecule. As used herein, the term “catalytic domain” refers to aprotein domain consisting of at least about 150-400, preferably about200-350, more preferably about 220-300, even more preferably at leastabout 240-280, and even more preferably about 260 amino acid residues inlength. In one embodiment, a MEKK catalytic domain contains at leastabout 9-13, preferably about 10-12, and more preferably about 11consensus kinase domains which are conserved among MEKK protein familymembers. Such consensus kinase domains are indicated by roman numeralsin FIG. 13. Particularly conserved residues within the consensus kinasedomains are underlined. A consensus kinase domain is further defined inHanks et al. (1988) Science 241:42-52. In another embodiment, a MEKKcatalytic domain is identified based in its ability to retain afunctional activity of a MEKK protein, particularly a MEKK protein(e.g., retains the ability to phosphorylate a MEKK substrate) even inthe absence of a MEKK regulatory domain, defined herein.

In another embodiment, a MEKK protein is identified based on thepresence of at least a “regulatory domain” in the protein orcorresponding nucleic acid molecule. As used herein, the term“regulatory domain” refers to a protein domain consisting of at leastabout 250-500, preferably about 300-450, more preferably about 320-400,even more preferably at least about 340-380, and even more preferablyabout 360 amino acid residues in length, of which at least 10%,preferably about 15%, and more preferably about 20% of the amino acidresidues are serine and/or threonone residues. In another embodiment, aMEKK regulatory domain is identified based on its ability to regulatethe activity of a MEKK catalytic domain. In one exemplary embodiment, aMEKK regulatory domain is capable of binding a MEKK binding partner suchthat the activity of a MEKK protein is modulated.

As used interchangeably herein, a “MEKK activity”, “functional activityof MEKK”, or “biological activity of MEKK”, refers to an activityexerted by a MEKK protein, polypeptide or nucleic acid molecule asdetermined in vivo, or in vitro, according to standard techniques. Inone embodiment, a MEKK activity is a direct activity, such as anassociation with a MEKK-target molecule. As used herein, a “targetmolecule” is a molecule with which a MEKK protein binds or interacts innature, such that MEKK-mediated function is achieved. A MEKK targetmolecule can be a MEKK protein or polypeptide of the present inventionor a non-MEKK molecule. For example, a MEKK target molecule can be anon-MEKK protein molecule (e.g., a MEKK binding partner such as a Rasprotein, or a MEKK substrate such as a MEK protein). As used herein, a“MEKK” substrate is a molecule with which a MEKK protein interacts invivo or in vitro such that the MEKK substrate is phosphorylated by theenzymatic activity of the MEKK protein. Also as used herein, a MEKK“binding partner” is a molecule with which a MEKK protein interacts invivo or in vitro such that the enzymatic activity of the MEKK protein iseffected. Alternatively, a MEKK activity is an indirect activity, suchas an activity mediated by interaction of the MEKK protein with a MEKKtarget molecule such that the target molecule modulates a downstreamcellular activity (e.g., MAPK activity).

In a preferred embodiment, a MEKK activity is at least one or more ofthe following activities: (i) interaction of a MEKK protein with a MEKKtarget molecule, wherein the target molecule effects the activity of theMEKK molecule; (ii) interaction of a MEKK protein with a MEKK targetmolecule, wherein the MEKK molecule effects the activity of the targetmolecule; (iii) phosphorylation of a MEKK target molecule (e.g., MEK orJNK kinase); (iv) activation of a MEKK target molecule (e.g., MEK or JNKkinase); (v) mediation of activation of MAPK signal transductionmolecules (e.g., c-Jun kinase (JNK) or p42/p44^(MAPK)); (vi)autophosphorylation of MEKK; (vii) autoactivation of MEKK 3; and (viii)modulation of the activity of a nuclear transcription factor (e.g., ATF2).

Accordingly, another embodiment of the invention features isolated MEKKproteins and polypeptides having a MEKK activity. Preferred proteins areMEKK proteins having at least a MEKK catalytic domain and, preferably, aMEKK activity. Additional preferred proteins are MEKK proteins having atleast a MEKK regulatory domain and, preferably, a MEKK activity. Inanother preferred embodiment, the isolated protein is a MEKK proteinhaving a MEKK catalytic domain, a MEKK regulatory domain, and a MEKKactivity.

As used herein, the term “antibody” is intended to includeimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site which specifically binds (immunoreacts with) an antigen,such as Fab and F(ab′)₂ fragments. The terms “monoclonal antibodies” and“monoclonal antibody composition”, as used herein, refer to a populationof antibody molecules that contain only one species of an antigenbinding site capable of immunoreacting with a particular epitope of anantigen, whereas the term “polyclonal antibodies” and “polyclonalantibody composition” refer to a population of antibody molecules thatcontain multiple species of antigen binding sites capable of interacingwith a particular antigen. A monoclonal antibody compositions thustypically display a single binding affinity for a particular antigenwith which it immunoreacts.

There is a known and definite correspondence between the amino acidsequence of a particular protein and the nucleotide sequences that cancode for the protein, as defined by the genetic code (shown below).Likewise, there is a known and definite correspondence between thenucleotide sequence of a particular nucleic acid molecule and the aminoacid sequence encoded by that nucleic acid molecule, as defined by thegenetic code.

GENETIC CODE Alanine (Ala, A) GCA, GCC, GCG, GCT Arginine (Arg, R) AGA,ACG, CGA, CGC, CGG, CGT Asparagine (Asn, N) AAC, AAT Aspartic acid (Asp,D) GAC, GAT Cysteine (Cys, C) TGC, TGT Glutamic acid (Glu, E) GAA, GAGGlutamine (Gln, Q) CAA, CAG Glycine (Gly, G) GGA, GGC, GGG, GGTHistidine (His, H) CAC, CAT Isoleucine (Ile, I) ATA, ATC, ATT Leucine(Leu, L) CTA, CTC, CTG, CTT, TTA, TTG Lysine (Lys, K) AAA, AAGMethionine (Met, M) ATG Phenylalanine (Phe, F) TTC, TTT Proline (Pro, P)CCA, CCC, CCG, CCT Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCTThreonine (Thr, T) ACA, ACC, ACG, ACT Tryptophan (Trp, W) TGG Tyrosine(Tyr, Y) TAC, TAT Valine (Val, V) GTA, GTC, GTG, GTT Termination signal(end) TAA, TAG, TGA

An important and well known feature of the genetic code is itsredundancy, whereby, for most of the amino acids used to make proteins,more than one coding nucleotide triplet may be employed (illustratedabove). Therefore, a number of different nucleotide sequences may codefor a given amino acid sequence. Such nucleotide sequences areconsidered functionally equivalent since they result in the productionof the same amino acid sequence in all organisms (although certainorganisms may translate some sequences more efficiently than they doothers). Moreover, occasionally, a methylated variant of a purine orpyrimidine may be found in a given nucleotide sequence. Suchmethylations do not affect the coding relationship between thetrinucleotide codon and the corresponding amino acid.

In view of the foregoing, the nucleotide sequence of a DNA or RNAmolecule coding for a human MEKK protein of the invention (or anyportion thereof) can be used to derive the human MEKK amino acidsequence, using the genetic code to translate the DNA or RNA moleculeinto an amino acid sequence. Likewise, for any human MEKK-amino acidsequence, corresponding nucleotide sequences that can encode the humanMEKK protein can be deduced from the genetic code (which, because of itsredundancy, will produce multiple nucleic acid sequences for any givenamino acid sequence). Thus, description and/or disclosure herein of ahuman MEKK nucleotide sequence should be considered to also includedescription and/or disclosure of the amino acid sequence encoded by thenucleotide sequence. Similarly, description and/or disclosure of a humanMEKK amino acid sequence herein should be considered to also includedescription and/or disclosure of all possible nucleotide sequences thatcan encode the amino acid sequence.

The human MEKK1 cDNA, which is approximately 3911 nucleotides in length,encodes a protein which is approximately 1302 amino acid residues inlength. The coding region is from nucleotide 3 to 3908 of SEQ ID NO:1.The human MEKK1 protein has at least a catalytic domain. A catalyticdomain includes, for example, about amino acids 1038-1302 of SEQ ID NO:2(e.gcatalytic domain having 259 or 260-265 amino acid residues).Catalytic domains having 100, 150, 200, or 250 consecutive amino acidsfrom about amino acids 1038-1302 of SEQ ID NO:2 are also intended to bewithin the scope of the invention. The human MEKK1 protein further hasat least a regulatory domain. A regulatory domain includes, for example,about amino acids xx-xxx of SEQ ID NO:2. Regulatory domains having 200,250, 300, or 350 consecutive amino acids from about amino acids xx-xxxof SEQ ID NO:2 are also intended to be within the scope of theinvention. Also intended to be within the scope of the invention aremodified catalytic and regulatory domains having about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35,40,45,50, 55, 60, 65,70,75. 80, 85, 90, 95, 100, 105, 110, 115, 116, 117, 118,119, 120, 121, 122, 123, 145, 125 amino acid substitutions, insertions,and/or deletions when compared to the amino acid sequence of SEQ IDNO:2, wherein the modified catalytic or regulatory domain retains thefunction of a MEKK catalytic or regualtory domain of SEQ ID NO:2.

The human MEKK2 cDNA, which is approximately 2013 nucleotides in length,encodes a protein which is approximately 619 amino acid residues inlength. The coding region is from nucleotide 124 to 1980 of SEQ ID NO:3.The human MEKK2 protein has at least a catalytic domain. A catalyticdomain includes, for example, about amino acids 361-619 of SEQ ID NO:4.Catalytic domains having 100, 150, 200, or 250 consecutive amino acidsfrom about amino acids 361-619 of SEQ ID NO:4 are also intended to bewithin the scope of the invention. The human MEKK2 protein further hasat least a regulatory domain. A regulatory domain includes, for example,about amino acids 1-360 of SEQ ID NO:4. Regulatory domains having 200,250, 300 or 350 consecutive amino acids from about amino acids 1-360 ofSEQ ID NO:4 are also intended to be within the scope of the invention.Also intended to be within the scope of the invention are modifiedcatalytic and regulatory domains having about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55,60, 61, or 62 amino acid substitutions, insertions, and/or deletionswhen compared to the amino acid sequence of SEQ ID NO:4, wherein themodified catalytic or regulatory domain retains the function of a MEKKcatalytic or regualtory domain of SEQ ID NO:4.

The human MEKK3 cDNA, which is approximately 1935 nucleotides in length,encodes a protein which is approximately 626 amino acid residues inlength. The coding region is from nucleotide 25 to 1902 of SEQ ID NO:5.The human MEKK3 protein has at least a catalytic domain. A catalyticdomain includes, for example, about amino acids 367-626 of SEQ ID NO:6.Catalytic domains having 100, 150, 200, or 250 consecutive amino acidsfrom about amino acids 367-626 of SEQ ID NO:6 are also intended to bewithin the scope of the invention. The human MEKK3 protein further hasat least a regulatory domain. A regulatory domain includes, for example,about amino acids 1-366 of SEQ ID NO:6. Regulatory domains having 200,250, 300, or 350 consecutive amino acids from about amino acids 1-366 ofSEQ ID NO:6 are also intended to be within the scope of the invention.Also intended to be within the scope of the invention are modifiedcatalytic and regulatory domains having about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40 amino acidsubstitutions, insertions, and/or deletions when compared to the aminoacid sequence of SEQ ID NO:6, wherein the modified catalytic orregulatory domain retains the function of a MEKK catalytic or regualtorydomain of SEQ ID NO:6.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode human MEKK proteins. The nucleotide sequence of human MEKK1,and corresponding predicted amino acid sequence, are shown in SEQ IDNODS:1 and 2, respectively. The nucleotide sequence of human MEKK2, andcorresponding predicted amino acid sequence, are shown in SEQ ID NODS:3and 4, respectively. The nucleotide sequence of human MEKK3, andcorresponding predicted amino acid sequence, are shown in SEQ ID NODS:5and 6, respectively. In a preferred embodiment, the nucleic acidmolecule comprises the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3,or SEQ ID NO:5. In other embodiments, the nucleic acid molecule has atleast 90-91% nucleotide identity, more preferably 92-93% nucleotideidentity, more preferably 94-95% nucleotide identity, more preferably96-97% nucleotide identity, more preferably 98-99% nucleotide identity,and even more preferably 99.5% nucleotide identity with the nucleotidesequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

Nucleic acid molecules that differ from SEQ ID NO:1, SEQ ID NO:3, or SEQID NO:5 due to degeneracy of the genetic code, and thus encode the samehuman MEKK protein as that encoded by SEQ ID NO:1, SEQ ID NO:3, or SEQID NO:5, are encompassed by the invention. Accordingly, in anotherembodiment, an isolated nucleic acid molecule of the invention has anucleotide sequence encoding a protein having an amino acid sequenceshown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.

A nucleic acid molecule having the nucleotide sequence of human MEKK1,human MEKK2, or human MEKK3 can be isolated using standard molecularbiology techniques and the sequence information provided herein. Forexample, a human MEKK DNA can be isolated from a human genomic DNAlibrary using all or portion of SEQ ID NO: 1, SEQ ID NO:3, or SEQ IDNO:5 as a hybridization probe and standard hybridization techniques(e.g., as described in Sambrook, J., et al. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989). Moreover, a nucleic acid molecule encompassing allor a portion of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 can be isolatedby the polymerase chain reaction using oligonucleotide primers designedbased upon the sequence of SEQ ID NO 1, SEQ ID NO:3, or SEQ ID NO:5. Forexample, mRNA can be isolated from cells (e.g., by theguanidinium-thiocyanate extraction procedure of Chirgwin et al. (1979)Biochemistry 18: 5294-5299) and cDNA can be prepared using reversetranscriptase (e.g., Moloney MLV reverse transcriptase, available fromGibco/BRL, Bethesda, Md.; or AMV reverse transcriptase, available fromSeikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for PCR amplification can be designed based uponthe nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, or SEQ IDNO:5. A nucleic acid of the invention can be amplified using cDNA or,alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to a human MEKK nucleotidesequence can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In addition to the human MEKK nucleotide sequence shown in SEQ ID NO:1,SEQ ID NO:3, or SEQ ID NO:5, it will be appreciated by those skilled inthe art that DNA sequence polymorphisms that lead to minor changes inthe nucleotide or amino acid sequences of human MEKK may exist within apopulation. Such genetic polymorphism in the human MEKK gene may existamong individuals within a population due to natural allelic variation.Such natural allelic variations can typically result in 1-2% variance inthe nucleotide sequence of the a gene. Any and all such nucleotidevariations and resulting amino acid polymorphisms in human MEKK that arethe result of natural allelic variation and that do not alter thefunctional activity of human MEKK are intended to be within the scope ofthe invention.

Nucleic acid molecules corresponding to natural allelic variants of thehuman MEKK DNAs of the invention can be isolated based on their homologyto the human MEKK nucleic acid molecules disclosed herein using thehuman DNA, or a portion thereof, as a hybridization probe according tostandard hybridization techniques under high stringency hybridizationconditions. Accordingly, in another embodiment, an isolated nucleic acidmolecule of the invention hybridizes under high stringency conditions toa second nucleic acid molecule comprising the nucleotide sequence of SEQID NO:1, SEQ ID NO:3, or SEQ ID NO:5. In certain embodiment, theisolated nucleic acid molecule comprises at least 30, 50, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 2000 or 3000 contiguous nucleotidesof SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. Preferably, an isolatednucleic acid molecule of the invention that hybridizes under highstringency conditions to the sequence of SEQ ID NO:1, SEQ ID NO:3, orSEQ ID NO:5 corresponds to a naturally-occurring allelic variant of ahuman MEKK nucleic acid molecule.

In addition to naturally-occurring allelic variants of the human MEKKsequence that may exist in the population, the skilled artisan willfurther appreciate that minor changes may be introduced by mutation intothe nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5,thereby leading to changes in the amino acid sequence of the encodedprotein, without altering the functional activity of the human MEKKprotein. For example, nucleotide substitutions leading to amino acidsubstitutions at “non-essential” amino acid residues may be made in thesequence of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5. A “non-essential”amino acid residue is a residue that can be altered from the wild-typesequence of human MEKK (e.g., the sequence of SEQ ID NO:2, SEQ ID NQ:4,or SEQ ID NO:6) without altering the functional activity of MEKK,whereas an “essential” amino acid residue is required for functionalactivity.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding human MEKK proteins that contain changes in aminoacid residues that are not essential for human MEKK activity. Such humanMEKK proteins differ in amino acid sequence from SEQ ID NO:2, SEQ IDNO:4, or SEQ ID NO:6 yet retain human MEKK activity. These non-naturalvariants of human MEKK also differ from non-human MEKK proteins (e.g.,mouse or rat MEKK) in that they encode at least one amino acid residuethat is unique to human MEKK (i.e., at least one residue that is notpresent in mouse or rat MEKK). Preferably, these non-natural variants ofhuman MEKK encode at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acidresidues that are unique to human MEKK (i.e., that are not present inmouse or rat MEKK).

An isolated nucleic acid molecule encoding a non-natural variant of ahuman MEKK protein can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequence ofSEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 (or plasmid pHu-MEKK) such thatone or more amino acid substitutions, additions or deletions areintroduced into the encoded protein. Mutations can be introduced intoSEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5 by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. Preferably,conservative amino acid substitutions are made at one or morenon-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art,including basic side chains (e.g., lysine, arginine, histidine), acidicside chains (e.g., aspartic acid, glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a nonessential amino acid residue in human MEKK ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of the human MEKK coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for their ability to bind to DNA and/or activate transcription,to identify mutants that retain functional activity. Followingmutagenesis, the encoded human MEKK mutant protein can be expressedrecombinantly in a host cell and the functional activity of the mutantprotein can be determined using assays available in the art forassessing MEKK activity (e.g., assays such as those described in detailin PCT Publication WO 97/39721 and/or asays described in Blank et al.(1996) J. Biol. Chem. 271:5361-5368.

Another aspect of the invention pertains to isolated nucleic acidmolecules that are antisense to the coding strand of a human MEKK MRNAor gene. An antisense nucleic acid of the invention can be complementaryto an entire human MEKK coding strand, or to only a portion thereof. Inone embodiment, an antisense nucleic acid molecule is antisense to acoding region of the coding strand of a nucleotide sequence encodinghuman MEKK that is unique to human MEKK (as compared to non-human MEKKs,such as mouse or rat MEKK). In another embodiment, the antisense nucleicacid molecule is antisense to a noncoding region of the coding strand ofa nucleotide sequence encoding human MEKK that is unique to human MEKK(as compared to non-human MEKKs, such as mouse or rat MEKK). Inpreferred embodiments, an antisense of the invention comprises at leastcontiguous nucleotides of the noncoding strand of SEQ ID NO:1, SEQ IDNO:3, or SEQ ID NO:5, more preferably at least 50, 100, 200, 300, 400,500, 600, 700, 800, 900 or 1000 contiguous nucleotides of the noncodingstrand of SEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5.

Given the coding strand sequences encoding human MEKK disclosed herein(e.g., nucleotides 3 to 3908 of SEQ ID NO:1, nucleotides 124-1980 of SEQID NO:3, or nucleotides 25-1902 of SEQ ID NO:5), antisense nucleic acidsof the invention can be designed according to the rules of Watson andCrick base pairing. The antisense nucleic acid molecule may becomplementary to the entire coding region of human MEKK mRNA, oralternatively can be an oligonucleotide which is antisense to only aportion of the coding or noncoding region of human MEKK mRNA. Forexample, the antisense oligonucleotide may be complementary to theregion surrounding the translation start site of human MEKK mRNA. Anantisense oligonucleotide can be, for example, about 15, 20, 25, 30, 35,40, 45 or 50 nucleotides in length. An antisense nucleic acid of theinvention can be constructed using chemical synthesis and enzymaticligation reactions using procedures known in the art. For example, anantisense nucleic acid (e.g., an antisense oligonucleotide) can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Alternatively, the antisense nucleic acid can be producedbiologically using an expression vector into which a nucleic acid hasbeen subcloned in an antisense orientation (i.e., RNA transcribed fromthe inserted nucleic acid will be of an antisense orientation to atarget nucleic acid of interest, described further in the followingsubsection).

In another embodiment, an antisense nucleic acid of the invention is aribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. A ribozymehaving specificity for a human MEKK-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a human MEKK genedisclosed herein. For example, a derivative of a Tetrahymena L-19 IVSRNA can be constructed in which the base sequence of the active site iscomplementary to the base sequence to be cleaved in a humanMEKK-encoding mRNA. See for example Cech et al. U.S. Pat. No. 4,987,071;and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, human MEKK mRNAcan be used to select a catalytic RNA having a specific ribonucleaseactivity from a pool of RNA molecules. See for example Bartel, D, andSzostak, J. W. (1993) Science 261: 1411-1418.

Yet another aspect of the invention pertains to isolated nucleic acidmolecules encoding human MEKK fusion proteins. Such nucleic acidmolecules, comprising at least a first nucleotide sequence encoding ahuman MEKK protein, polypeptide or peptide operatively linked to asecond nucleotide sequence encoding a non-human MEKK protein,polypeptide or peptide, can be prepared by standard recombinant DNAtechniques. Human MEKK fusion proteins are described in further detailbelow in subsection III.

II. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyrecombinant expression vectors, containing a nucleic acid encoding humanMEKK (or a portion thereof). The expression vectors of the inventioncomprise a nucleic acid of the invention in a form suitable forexpression of the nucleic acid in a host cell, which means that therecombinant expression vectors include one or more regulatory sequences,selected on the basis of the host cells to be used for expression, whichis operatively linked to the nucleic acid sequence to be expressed.Within a recombinant expression vector, “operably linked” is intended tomean that the nucleotide sequence of interest is linked to theregulatory sequence(s) in a manner which allows for expression of thenucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell). The term “regulatory sequence” is intended to includes promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel; Gene Expression Technology. Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Regulatory sequences include thosewhich direct constitutive expression of a nucleotide sequence in manytypes of host cell and those which direct expression of the nucleotidesequence only in certain host cells (e.g., tissue-specific regulatorysequences). It will be appreciated by those skilled in the art that thedesign of the expression vector may depend on such factors as the choiceof the host cell to be transformed, the level of expression of proteindesired, etc. The expression vectors of the invention can be introducedinto host cells to thereby produce proteins or peptides, includingfusion proteins or peptides, encoded by nucleic acids as describedherein (e.g., human MEKK proteins, mutant forms of human MEKK proteins,human MEKK fusion proteins and the like).

The recombinant expression vectors of the invention can be designed forexpression of human MEKK protein in prokaryotic or eukaryotic cells. Forexample, human MEKK can be expressed in bacterial cells such as E. coli,insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector may be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotorsdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors can serve one or more purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification; 4) to provide an epitopetag to aid in detection and/or purification of the protein; and/or 5) toprovide a marker to aid in detection of the protein (e.g., a colormarker using β-galactosidase fusions). Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc.;Smith, D. B, and Johnson, K. S. (1988) Gene 67:3140), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. Recombinantproteins also can be expressed in eukaryotic cells as fusion proteinsfor the same purposes discussed above.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada e1 al., (1992) Nuc. Acids Res.20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the human MEKK expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerivisae include pYepSec1 (Baldari. et al., (1987) EMBO J. 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal., (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

Alternatively, human MEKK can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf 9 cells)include the pAc series (Smith et al., (1983) Mol. Cell Biol.3:2156-2165) and the pVL series (Lucklow, V. A., and Summers, M. D.,(1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pMex-NeoI, pCDM8 (Seed, B., (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements.

For example, commonly used promoters are derived from polyoma,Adenovirus 2, cytomegalovirus and Simian Virus 40.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include lymphoid-specific promoters (Calame and Eaton (1988)Adv. Immunol. 43:235-275), in particular promoters of T cell receptors(Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins(Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell33:741-748), the albumin promoter (liver-specific; Pinkert et al (1987)Genes Dev. 1:268-277), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

Moreover, inducible regulatory systems for use in mammalian cells areknown in the art, for example systems in which gene expression isregulated by heavy metal ions (see e.g., Mayo et al. (1982) Cell29:99-108; Brinster et al. (1982) Nature 296:39-42; Searle et al. (1985)Mol. Cell. Biol. 5:1480-1489), heat shock (see e.g., Nouer et al. (1991)in Heat Shock Response, e.d. Nouer, L. , CRC, Boca Raton, Fla.,pp167-220), hormones (see e.g., Lee et al. (1981) Nature 294:228-232;Hynes et al. (1981) Proc. Natl. Acad. Sci. USA 78:2038-2042; Klock etal. (1987) Nature 329:734-736; Israel & Kaufman (1989) Nucl. Acids Res.17:2589-2604; and PCT Publication No. WO 93/23431), FK506-relatedmolecules (see e.g., PCT Publication No. WO 94/18317) or tetracyclines(Gossen, M, and Bujard, H. (1992) Proc. Natl. Acad Sci. USA89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; PCTPublication No. WO 94/29442; and PCT Publication No. WO 96/01313).Accordingly, in another embodiment, the invention provides a recombinantexpression vector in which human MEKK DNA is operatively linked to aninducible eukaryotic promoter, thereby allowing for inducible expressionof human MEKK protein in eukaryotic cells.

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a marner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to human MEKK mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Antisense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to recombinant host cells intowhich a vector, preferably a recombinant expression vector, of theinvention has been introduced. A host cell may be any prokaryotic oreukaryotic cell. For example. human MEKK protein may be expressed inbacterial cells such as E. coli, insect cells, yeast or mammalian cells(such as Chinese hamster ovary cells (CHO) or COS cells). Other suitablehost cells are known to those skilled in the art. Vector DNA can beintroduced into prokaryotic or eukaryotic cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in Sambrook et al. (Molecular Cloning: A LaboratoryManual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), andother laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker may be introduced into a host cell on the samevector as that encoding human MEKK or may be introduced on a separatevector. Cells stably transfected with the introduced nucleic acid can beidentified by drug selection (e.g., cells that have incorporated theselectable marker gene will survive, while the other cells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) human MEKKprotein. Accordingly, the invention firther provides methods forproducing human MEKK protein using the host cells of the invention. Inone embodiment, the method comprises culturing the host cell ofinvention (into which a recombinant expression vector encoding humanMEKK has been introduced) in a suitable medium until human MEKK isproduced. In another embodiment, the method further comprises isolatinghuman MEKK from the medium or the host cell. In its native form thehuman MEKK protein is an intracellular protein and, accordingly,recombinant human MEKK protein can be expressed intracellularly in arecombinant host cell and then isolated from the host cell, e.g., bylysing the host cell and recovering the recombinant human MEKK proteinfrom the lysate. Alternatively, recombinant human MEKK protein can beprepared as a extracellular protein by operatively linking aheterologous signal sequence to the amino-terminus of the protein suchthat the protein is secreted from the host cells. In this case,recombinant human MEKK protein can be recovered from the culture mediumin which the cells are cultured.

Certain host cells of the invention can also be used to produce nonhumantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichhuman MEKK-coding sequences have been introduced. Such host cells canthen be used to create non-human transgenic animals in which exogenoushuman MEKK sequences have been introduced into their genome orhomologous recombinant animals in which endogenous MEKK sequences havebeen altered. Such animals are useful for studying the function and/oractivity of human MEKK and for identifying and/or evaluating modulatorsof human MEKK activity. Accordingly, another aspect of the inventionpertains to nonhuman transgenic animals which contain cells carrying atransgene encoding a human MEKK protein or a portion of a human MEKKprotein. In a subembodiment, of the transgenic animals of the invention,the transgene alters an endogenous gene encoding an endogenous MEKKprotein (e.g., homologous recombinant animals in which the endogenousMEKK gene has been functionally disrupted or “knocked out”, or thenucleotide sequence of the endogenous MEKK gene has been mutated or thetranscriptional regulatory region of the endogenous MEKK gene has beenaltered).

A transgenic animal of the invention can be created by introducing humanMEKK-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, and allowing the oocyte to develop in apseudopregnant female foster animal. The human MEKK nucleotide sequenceof SEQ ID NO:1 (and plasmid pHu-MEKK) can be introduced as a transgeneinto the genome of a non-human animal.

Intronic sequences and polyadenylation signals can also be included inthe transgene to increase the efficiency of expression of the transgene.A tissue-specific regulatory sequence(s) can be operably linked to thehuman MEKK transgene to direct expression of human MEKK protein toparticular cells. Methods for generating transgenic animals via embryomanipulation and microinjection, particularly animals such as mice, havebecome conventional in the art and are described, for example, in U.S.Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al, and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the human MEKK transgene in its genome and/or expression ofhuman MEKK mRNA in tissues or cells of the animals. A transgenic founderanimal can then be used to breed additional animals carrying thetransgene. Moreover, transgenic animals carrying a transgene encodinghuman MEKK can further be bred to other transgenic animals carryingother transgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a human MEKK gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the endogenous MEKK gene. In one embodiment, ahomologous recombination vector is designed such that, upon homologousrecombination, the endogenous MEKK gene is functionally disrupted (i.e.,no longer encodes a functional protein; also referred to as a “knockout” vector). Alternatively, the vector can be designed such that, uponhomologous recombination, the endogenous MEKK gene replaced by the humanMEKK gene. In the homologous recombination vector, the altered portionof theMEKK gene is flanked at its 5′ and 3′ ends by additional nucleicacid of the MEKK gene to allow for homologous recombination to occurbetween the exogenous human MEKK gene carried by the vector and anendogenous MEKK gene in an embryonic stem cell. The additional flankingMEKK nucleic acid is of sufficient length for successful homologousrecombination with the endogenous gene. Typically, several kilobases offlanking DNA (both at the 5′ and 3′ ends) are included in the vector(see e.g., Thomas, K. R, and Capecchi, M. R. (1987) Cell 51:503 for adescription of homologous recombination vectors). The vector isintroduced into an embryonic stem cell line (e.g., by electroporation)and cells in which the introduced human MEKK gene has homologouslyrecombined with the endogenous MEKK gene are selected (see e.g., Li, E.et al. (1992) Cell 69:915). The selected cells are then injected into ablastocyst of an animal (e.g., a mouse) to form aggregation chimeras(see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: APractical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp.113-152). A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term.Progeny harboring the homologously recombined DNA in their germ cellscan be used to breed animals in which all cells of the animal containthe homologously recombined DNA by germline transmission of thetransgene. Methods for constructing homologous recombination vectors andhomologous recombinant animals are described f1irther in Bradley, A.(1991) Current Opinion in Biotechnology 2:823-829 and in PCTInternational Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO93/04169 by Berns et al.

In addition to the foregoing, the skilled artisan will appreciate thatother approaches known in the art for homologous recombination can beapplied to the instant invention. Enzyme-assisted site-specificintegration systems are known in the art and can be applied to integratea DNA molecule at a predetermined location in a second target DNAmolecule. Examples of such enzyme-assisted integration systems includethe Cre recombinase-lox target system (e.g., as described in Baubonis,W, and Sauer, B. (1993) Nucl. Acids Res. 21:2025-2029; and Fukushige, S,and Sauer, B. (1992) Proc. Natl. Acad. Sci. USA 89:7905-7909) and theFLP recombinase-FRT target system (e.g., as described in Dang, D. T, andPerrimon, N. (1992) Dev. Genet. 13:367-375; and Fiering, S. et al.(1993) Proc. Natl. Acad. Sci. USA 90:8469-8473). Tetracycline-regulatedinducible homologous recombination systems, such as described in PCTPublication No. WO 94/29442 and PCT Publication No. WO 96/01313, alsocan be used.

III. Isolated Human MEKK Proteins and Anti-Human MEKK Antibodies

Another aspect of the invention pertains to isolated human MEKKproteins. Preferably, the human MEKK protein comprises the amino acidsequence of SEQ ID NO: 2. In other embodiments, the protein has at least90-91% amino acid identity, more preferably 92-93% amino identity, morepreferably 94-95% amino identity, more preferably 96-97% amino identity,more preferably 98-99% amino identity, and even more preferably 99.5%amino acid identity with the amino acid sequence of SEQ ID NO:2, SEQ IDNO:4, or SEQ ID NO:6.

In other embodiments, the invention provides isolated portions of thehuman MEKK protein. For example, the invention further encompasses anamino-terninal portion of human MEKK that includes a regulatory domain.This portion encompasses, for example, about amino acids l-xxx of SEQ IDNO:2, about amino aicds 1-360 of SEQ ID NO:4, or about amino acids 1-366of SEQ ID NO:6. Another isolated portion of human MEKK provided by theinvention is a carboxy-terminal catalytic domain. This portionencompasses, for example, about amino acids 1038-1302 of SEQ ID NO:2,about amino acids 361-619 of SEQ ID NO:4, or about amino acids 367-626of SEQ ID NO:6. In yet other embodiments, the invention providesbiologically active portions of the human MEKK protein.

As used interchangeably herein, a “MEKK activity”, “biological activityof MEKK” or “functional activity of MEKK”, refers to an activity exertedby a MEKK protein, polypeptide or portion thereof as determined in vivo,or in vitro, according to standard techniques.

In one embodiment, a MEKK activity is a direct activity, such as anassociation with a MEKK-target molecule. As used herein, a “targetmolecule” is a molecule with which a MEKK protein binds or interacts innature, such that MEKK-mediated function is acheived. A MEKK targetmolecule can be a non-MEKK molecule or a MEKK protein or polypeptide ofthe present invention (e.g., an autoactivity). In an exemplaryembodiment, a MEKK target molecule is a MEKK substrate (e.g., MEK orJNKK). Alternatively, an STMST activity is an indirect activity, such asa cellular signaling activity mediated by interaction of the MEKKprotein with a MEKK ligand.

In a preferred embodiment, a MEKK activity is at least one or more ofthe following activities: (i) interaction of a MEKK protein with solubleMEKK ligand (e.g., MEK or JNKK); (ii) modulation of the activity of aMEKK substrate; (iii) activation of a MEKK substrate; (iv) indirectmodulation of a downstream signaling molecule (e.g., MAPK, for examplep⁴²/p^(44MAPK) or (JNK).

In yet another preferred embodiment, a MEKK activity is at least one ormore of the following activities: (1) modulation of cellular signaltransduction, either in vitro or in vivo; (2) regulation of genetranscription in a cell expressing a MEKK protein; (3) regulation ofgene transcription in a cell expressing a MEKK protein, wherein saidcell is involved inflammation; (4) regulation of cellular proliferation;(5) regulation of cellular differentiation; (6) regulation ofdevelpoment; (7) regulation of cell death; or (8) regulation ofregulation of inflammation.

Human MEKK proteins of the invention are preferably produced byrecombinant DNA techniques. For example, a nucleic acid moleculeencoding the protein is cloned into an expression vector (as describedabove), the expression vector is introduced into a host cell (asdescribed above) and the human MEKK protein is expressed in the hostcell. The human MEKK protein can then be isolated from the cells by anappropriate purification scheme using standard protein purificationtechniques. Alternative to recombinant expression, a human MEKKpolypeptide can be synthesized chemically using standard peptidesynthesis techniques. Moreover, native human MEKK protein can beisolated from cells (e.g., from T cells), for example byimmunoprecipitation using an anti-human MEKK antibody.

The invention also provides human MEKK fusion proteins. As used herein,a human MEKK “fusion protein” comprises a human MEKK polypeptideoperatively linked to a polypeptide other than human MEKK. A “human MEKKpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to human MEKK protein, or a peptide fragment thereof whichis unique to human MEKK protein (as compared to non-human MEKK proteins,such as mouse or chicken MEKK”, whereas a “polypeptide other than humanMEKK” refers to a polypeptide having an amino acid sequencecorresponding to another protein. Within the fusion protein, the term“operatively linked” is intended to indicate that the human MEKKpolypeptide and the other polypeptide are fused in-frame to each other.The other polypeptide may be fi1ed to the N-tenninus or C-terminus ofthe human MEKK polypeptide. For example, in one embodiment, the fusionprotein is a GST-human MEKK fusion protein in which the human MEKKsequences are fused to the C-terminus of the GST sequences. In anotherembodiment, the fusion protein is a human MEKK-HA fusion protein inwhich the human MEKK nucleotide sequence is inserted in a vector such aspCEP4-HA vector (Herrscher, R. F. et al. (1995) Genes Dev. 9:3067-3082)such that the human MEKK sequences are fused in frame to an influenzahemagglutinin epitope tag. Such fusion proteins can facilitate thepurification of recombinant human MEKK.

Preferably, a human MEKK fusion protein of the invention is produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor the different polypeptide sequences are ligated together in-frame inaccordance with conventional techniques, for example employingblunt-ended or stagger-ended termini for ligation, restriction enzymedigestion to provide for appropriate termini, filling-in of cohesiveends as appropriate, alkaline phosphatase treatment to avoid undesirablejoining, and enzymatic ligation. In another embodiment, the fusion genecan be synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and reamplified to generate a chimeric gene sequence (see,for example, Current Protocols in Molecular Biology, eds. Ausubel et al.John Wiley & Sons: 1992). Moreover, many expression vectors arecommercially available that already encode a fusion moiety (e.g., a GSTpolypeptide or an HA epitope tag). A human MEKK-encoding nucleic acidcan be cloned into such an expression vector such that the fusion moietyis linked in-frame to the human MEKK protein.

An isolated human MEKK protein, or fragment thereof, can be used as animmunogen to generate antibodies that bind specifically to human MEKKusing standard techniques for polyclonal and monoclonal antibodypreparation. The human MEKK protein can be used to generate antibodiesor, alternatively, an antigenic peptide fragment of human MEKK can beused as the immunogen. An antigenic peptide fragment of human MEKKtypically comprises at least 8 amino acid residues of the amino acidsequence shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 andencompasses an epitope of human MEKK such that an antibody raisedagainst the peptide forms a specific immune complex with human MEKK.Preferably, the antigenic peptide comprises at least 10 amino acidresidues, more preferably at least 15 amino acid residues, even morepreferably at least 20 amino acid residues, and most preferably at least30 amino acid residues. Preferred epitopes encompassed by the antigenicpeptide are regions of human MEKK that are located on the surface of theprotein, e.g., hydrophilic regions, and that are unique to human MEKK,as compared to MEKK proteins from other species, such as chicken ormouse (ie., an antigenic peptide that spans a region of human MEKK thatis not conserved across species is used as immunogen; such non-conservedregions/residues are underlined and bolded in FIG. 4, FIG. 8, or FIG.12). A standard hydrophobicity analysis of the human MEKK protein can beperformed to identify hydrophilic regions.

A human MEKK immunogen typically is used to prepare antibodies byimmunizing a suitable subject, (e.g., rabbit, goat, mouse or othermammal) with the immunogen. An appropriate immunogenic preparation cancontain, for examples, recombinantly expressed human MEKK protein or achemically synthesized human MEKK peptide. The preparation can furtherinclude an adjuvant, such as Freund's complete or incomplete adjuvant,or similar immunostimulatory agent. Immunization of a suitable subjectwith an immunogenic human MEKK preparation induces a polyclonalanti-human MEKK antibody response.

Accordingly, another aspect of the invention pertains to anti-human MEKKantibodies. Polyclonal anti-human MEKK antibodies can be prepared asdescribed above by immunizing a suitable subject with a human MEKKimmunogen. The anti-human MEKK antibody titer in the immunized subjectcan be monitored over time by standard techniques, such as with anenzyme linked immunosorbent assay (ELISA) using immobilized human MEKK.If desired, the antibody molecules directed against human MEKK can beisolated from the mammal (e.g., from the blood) and further purified bywell known techniques, such as protein A chromatography to obtain theIgG fraction. At an appropriate time after immunization, e.g., when theanti-human MEKK antibody titers are highest, antibody-producing cellscan be obtained from the subject and used to prepare monoclonalantibodies by standard techniques, such as the hybridoma techniqueoriginally described by Kohler and Milstein (1975, Nature 256:495-497)(see also, Brown et al. (1981) J. Immunol 127:539-46; Brown et al.(1980) J Biol Chem 255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; andYeh et al. (1982) Int. J Cancer 29:269-75), the more recent human B cellhybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), theEBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies andCancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. Thetechnology for producing monoclonal antibody hybridomas is well known(see generally R. H. Kenneth, in Monoclonal Antibodies: A New DimensionIn Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980);E. A. Lerner (1981) Yale J. Biol. Med., 54:387402; M. L. Gefter et al.(1977) Somatic Cell Genet., 3:231-36). Briefly, an immortal cell line(typically a myeloma) is fused to lymphocytes (typically splenocytes)from a mammal immunized with a human MEKK immunogen as described above,and the culture supernatants of the resulting hybridoma cells arescreened to identify a hybridoma producing a monoclonal antibody thatbinds specifically to human MEKK.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-human MEKK monoclonal antibody (see, e.g., G. Galfre et al. (1977)Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lemer,Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, citedsupra). Moreover, the ordinary skilled worker will appreciate that thereare many variations of such methods which also would be useful.Typically, the immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. For example, murinehybridomas can be made by fusing lymphocytes from a mouse immunized withan immunogenic preparation of the present invention with an immortalizedmouse cell line. Preferred immortal cell lines are mouse myeloma celllines that are sensitive to culture medium containing hypoxanthine,aminopterin and thymidine (“HAT medium”). Any of a number of myelomacell lines may be used as a fusion partner according to standardtechniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14myeloma lines. These myeloma lines are available from the American TypeCulture Collection (ATCC), Rockville, Md. Typically, HAT-sensitive mousemyeloma cells are fused to mouse splenocytes using polyethylene glycol(“PEG”). Hybridoma cells resulting from the fusion are then selectedusing HAT medium, which kills unfused and unproductively fused myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridoma cells producing a monoclonal antibody of theinvention are detected by screening the hybridoma culture supernatantsfor antibodies that bind human MEKK, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-human MEKK antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with human MEKK to thereby isolateimmunoglobulin library members that bind human MEKK. Kits for generatingand screening phage display libraries are commercially available (e.g.,the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01;and the Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al.International Publication No. WO 92/18619; Dower et al. InternationalPublication No. WO 91/17271; Winter et al. International Publication WO92/20791; Markland et al. International Publication No. WO 92/15679;Breitling et al. International Publication WO 93/01288; McCafferty etal. International Publication No. WO 92/01047; Garrard et al.International Publication No. WO 92/09690; Ladner et al. InternationalPublication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse etal. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clarkson etal. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580;Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al.(1991) Nuc Acid Res 19:4133-4137; Barbas et al. (1991) PNAS88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

Additionally, recombinant anti-human MEKK antibodies, such as chimericand humanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Patent Publication PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT Application WO 86/01533; Cabilly et al. U.S. Pat. No.4,816,567; Cabilly et al. European Patent Application 125,023; Better etal. (1988) Science 240:1041-1043; Liu et al. (1987) PNAS 84:3439-3443;Liu et al. (1987) J Immunol. 139:3521-3526; Sun et al. (1987) PNAS84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al.(1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

An anti-human MEKK antibody (e.g., monoclonal antibody) can be used toisolate human MEKK by standard techniques, such as affinitychromatography or immunoprecipitation. An anti-human MEKK antibody canfacilitate the purification of natural human MEKK from cells and ofrecombinantly produced human MEKK expressed in host cells. Moreover, ananti-human MEKK antibody can be used to detect human MEKK protein (e.g.,in a cellular lysate or cell supernatant). Detection may be facilitatedby coupling (i.e., physically linking) the antibody to a detectablesubstance. Accordingly, in one embodiment, an anti-human MEKK antibodyof the invention is labeled with a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials and radioactive materials.Examples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Yet another aspect of the invention pertains to anti-human MEKKantibodies that are obtainable by a process comprising:

(a) immunizing an animal with an immunogenic human MEKK protein, or animmunogenic portion thereof unique to human MEKK protein; and

(b) isolating from the animal antibodies that specifically bind to ahuman MEKK protein.

Methods for immunization and recovery of the specific anti-human MEKKantibodies are described further above.

IV. Pharmaceutical Compositions

Human MEKK modulators of the invention (e.g., human MEKK inhibitory orstimulatory agents, including human MEKK proteins and antibodies) can beincorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the modulatoryagent and a pharmaceutically acceptable carrier. As used herein the term“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible ith its intended route of administration. For example,solutions or suspensions used for arenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These may be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

V. Methods of the Invention

A. Detection Assays

Another aspect of the invention pertains to methods of using the varioushuman MEKK compositions of the invention. For example, the inventionprovides a method for detecting the presence of human MEKK activity in abiological sample. The method involves contacting the biological samplewith an agent capable of detecting human MEKK activity, such as humanMEKK protein or human MEKK mRNA, such that the presence of human MEKKactivity is detected in the biological sample.

A preferred agent for detecting human MEKK mRNA is a labeled nucleicacid probe capable of specifically hybridizing to human MEKK mRNA. Thenucleic acid probe can be, for example, the human MEKK DNA of SEQ IDNO:1, SEQ ID NO:3, or SEQ ID NO:5 (or plasmid pHu-MEKK1, plasmidpHu-MEKK2, or plasmid pHu-MEKK3), or a portion thereof unique to humanMEKK (as compared to MEKK from other species, such as chicken or mouse),such as an oligonucleotide of at least 15, 30, 50, 100, 200, 300, 400,500, 600, 700, 800, 900 or 1000 nucleotides in length and sufficient tospecifically hybridize under stringent conditions to human MEKK mRNA.

A preferred agent for detecting human MEKK protein is a labeled antibodycapable of binding to human MEKK protein. Antibodies can be polyclonal,or more preferably, monoclonal. An intact antibody, or a fragmentthereof (e.g., Fab or F(ab′)₂) can be used. The term “labeled”, withregard to the probe or antibody, is intended to encompass directlabeling of the probe or antibody by coupling (ie., physically linking)a detectable substance to the probe or antibody, as well as indirectlabeling of the probe or antibody by reactivity with another reagentthat is directly labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently labeled secondaryantibody and end-labeling of a DNA probe with biotin such that it can bedetected with fluorescently labeled streptavidin. The term “biologicalsample” is intended to include tissues, cells and biological fluids. Forexample, techniques for detection of human MEKK mRNA include Northernhybridizations and in situ hybridizations. Techniques for detection ofhuman MEKK protein include enzyme linked immunosorbent assays (ELISAs),Western blots, immunoprecipitations and immunofluorescence.

B. Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, ie., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to MEKK proteins, have a stimulatory or inhibitory effect on,for example, MEKK expression or MEKK activity, or have a stimulatory orinhibitory effect on, for example, the activity of an MEKK targetmolecule.

In one embodiment, the invention provides assays for screening candidateor test compounds which bind to or interact with a MEKK protein orpolypeptide or biologically active portion thereof. In anotherembodiment, the invention provides assays for screening candidate ortest compounds which bind to or modulate the activity of a MEKK proteinor polypeptide or biologically active portion thereof. The testcompounds of the present invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the ‘one-bead one-compound’ library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des.12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In one embodiment, an assay is a cell-free assay for identifyingcompounds which bind to or interact with a MEKK protein of the presentinvention. For example, the invention provides a method for identifyinga compound that binds to or interacts with a human MEKK protein,comprising

providing an indicator composition that comprises a human MEKK protein,or biologically active portion thereof;

contacting the indicator composition with a test compound; and

determining the ability of the test compound to bind to or interact withthe human MEKK protein or biologically active portion thereof in theindicator composition to thereby identify a compound that binds to orinteracts with a human MEKK protein.

Determining the ability of the test compound to bind to or interact withthe MEKK protein or biologically active potion thereof can beaccomplished, for example, by coupling either the test compound or theMEKK protein or biologically active portion thereof with a radioisotopeor enzymatic label such that binding can be determined by detecting thelabeled test compound or MEKK protein or biologically active portionthereof in a complex. For example, compounds (e.g., MEKK protein orbiologically active portion thereof) can be labeled with ¹²⁵I, ³⁵S, ¹⁴C,or ³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemmission or by scintillation counting.Alternatively, compounds can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product.

Determining the ability of the test compound to bind to a MEKK proteinor biologically active portion thereof can also be accomplished using atechnology such as real-time Biomolecular Interaction Analysis (BIA).Sjolander, S, and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 andSzabo et al. (1995) Curr. Opin. Struct Biol. 5;699-705, As used herein,“BIA” is a technology for studying biospecific interactions in realtime, without labeling any of the interactants (e.g., BlAcore). Changesin the optical phenomenon of surface plasmon resonance (SPR) can be usedas an indication of real-time reactions between biological molecules.

In another embodiment, an assay of the present invention the inventionprovides a method for identifying a compound that modulates the activityof a human MEKK protein, comprising

providing an indicator composition that comprises a human MEKK protein,or biologically active portion thereof;

contacting the indicator composition with a test compound; and

determining the effect of the test compound on the activity of the humanMEKK protein or biologically active portion thereof, in the indicatorcomposition to thereby identify a compound that modulates the activityof a human MEKK protein.

Determining the effect of the test compound on the activity of the humanMEKK protein can be accomplished directly by detecting a biologicalactivity of the MEKK protein or portion thereof. Alternatively,determining the effect of the test compound on the activity of the humanMEKK protein can be accomplished by detecting activity of a downstreamtarget of MEKK, e.g., induction of a cellular second messenger of thetarget (i.e. intracellular Ca²⁺, diacylglycerol, IP₃, etc.),catalytic/enzymatic activity of the target an appropriate substrate,detecting the induction of a reporter gene (comprising atarget-responsive regulatory element operatively linked to a nucleicacid encoding a detectable marker, e.g., luciferase), or detecting atarget-regulated cellular response, for example, DNA:protein orprotein:protein interactions.

In another embodiment, a screening assay of the invention is a cellbased assay. For example, the indicator composition can comprise anindicator cell (e.g., a marnmalain cell or a yeast cell), wherein saidindicator cell comprises: (i) the a human MEKK protein or biologicallyactive portion thereof. Preferably, the indicator cell contains:

i) a recombinant expression vector encoding the human MEKK; and saidmethod comprises:

a) contacting the indicator cell with a test compound;

b) determining the effect of the test compound on the activity of theMEKK protein or biologically active portion thereof to thereby identifya compound that modulates the activity of human MEKK.

In another embodiment, the assay further comprises the step of

c) comparing the activity of the MEKK protein or biologically activeportion thereof in the indicator cell in the presence of the testcompound with the activity of the MEKK protein or biologically activeportion thereof in the indicator cell in the absence of the testcompound to thereby identify a compound that modulates the activity ofhuman MEKK.

In another example, the indicator composition can comprise an indicatorcell, wherein said indicator cell comprises: (i) the a human MEKKprotein or biologically active portion thereof and (ii) a reporter generesponsive to the human MEKK protein. Preferably, the indicator cellcontains:

i) a recombinant expression vector encoding the human MEKK; and

ii) a vector comprising regulatory sequences of a gene responsive toMEKK signal transduction (e,g, a gene containing regulatory sequencesresponsive to the transcription factor, ATF 2) operatively linked to areporter gene; and said method comprises:

a) contacting the indicator cell with a test compound;

b) determining the level of expression of the reporter gene in theindicator cell in the presence of the test compound to thereby identifya compound that modulates the activity of human MEKK.

In another embodiment, the assay further comprises the step of

c) comparing the level of expression of the reporter gene in theindicator cell in the presence of the test compound with the level ofexpression of the reporter gene in the indicator cell in the absence ofthe test compound to thereby identify a compound that modulates theactivity of human MEKK.

In another preferred embodiment, the indicator composition comprises apreparation of: (i) a human MEKK protein and (ii) a DNA molecule towhich an ATF 2 transcription factor binds, and

said method comprises:

a) contacting the indicator composition with a test compound;

b) determining the degree of interaction of an ATF 2 transcriptionfactor and the DNA molecule in the presence of the test compound; and

c) comparing the degree of interaction of ATF 2 transcription factor andthe DNA molecule in the presence of the test compound with the degree ofinteraction of the ATF 2 transcription factor and the DNA molecule inthe absence of the test compound to thereby identify a compound thatmodulates the activity of human MEKK.

In another preferred embodiment, the method identifies proteins thatinteract with human MEKK. In this embodiment,

the indicator composition is an indicator cell, which indicator cellcomprises:

i) a reporter gene operably linked to a transcriptional regulatorysequence; and

ii) a first chimeric gene which encodes a first fusion protein, saidfirst fusion protein including human MEKK;

the test compound comprises a library of second chimeric genes, whichlibrary encodes second fusion proteins;

expression of the reporter gene being sensitive to interactions betweenthe first fusion protein, the second fusion protein and thetranscriptional regulatory sequence; and

wherein the effect of the test compound on human MEKK in the indicatorcomposition is determined by detecting the level of expression of thereporter gene in the indicator cell to thereby identify a test compoundcomprising a protein that interacts with human MEKK.

Furthermore, the present invention provides assays comprising the stepof contacting an indicator composition with a compound which is known tointeract with, bind to, or modulate the activity of a MEKK protein orbiologically active portion thereof and determining the ability of atest compound to effect the ability of the known compound to bind to,interact with, or modulate the activity of the MEKK protein orbiologically active portion thereof.

Recombinant expression vectors that can be used for expression of humanMEKK in the indicator cell are known in the art (see discussions above).In one embodiment, within the expression vector the human MEKK-codingsequences are operatively linked to regulatory sequences that allow forconstitutive expression of human MEKK in the indicator cell (e.g., viralregulatory sequences, such as a cytomegalovirus promoter/enhancer, canbe used). Use of a recombinant expression vector that allows forconstitutive expression of human MEKK in the indicator cell is preferredfor identification of compounds that enhance or inhibit the activity ofhuman MEKK. In an alternative embodiment, within the expression vectorthe human MEKK-coding sequences are operatively linked to regulatorysequences of the endogenous human MEKK gene (i.e., the promoterregulatory region derived from the endogenous human MEKK gene). Use of arecombinant expression vector in which human MEKK expression iscontrolled by the endogenous regulatory sequences is preferred foridentification of compounds that enhance or inhibit the transcriptionalexpression of human MEKK.

A variety of reporter genes are known in the art and are suitable foruse in the screening assays of the invention. Examples of suitablereporter genes include those which encode chloramphenicolacetyltransferase, beta-galactosidase, alkaline phosphatase orluciferase. Standard methods for measuring the activity of these geneproducts are known in the art. Likewise, a variety of cell types aresuitable for use as an indicator cell in the screening assay. Preferablya cell line is used which does not normally express human MEKK.Mammalian cell lines as well as yeast cells can be used as indicatorcells.

In one embodiment, the level of expression of the reporter gene in theindicator cell in the presence of the test compound is higher than thelevel of expression of the reporter gene in the indicator cell in theabsence of the test compound and the test compound is identified as acompound that stimulates the expression or activity of human MEKK. Inanother embodiment, the level of expression of the reporter gene in theindicator cell in the presence of the test compound is lower than thelevel of expression of the reporter gene in the indicator cell in theabsence of the test compound and the test compound is identified as acompound that inhibits the expression or activity of human MEKK.

Alternative to the use of a reporter gene construct, compounds thatmodulate the expression or activity of human MEKK can be identified byusing other “read-outs.” For example, an indicator cell can betransfected with a human MEKK expression vector. incubated in thepresence and in the absence of a test compound, and MEKK activity beassessed by detecting the mRNA of an ATF 2-responsive gene product.Standard methods for detecting mRNA, such as reversetranscription-polymerase chain reaction (RT-PCR) are known in the art.Alternatively, MEKK activity can be assesed by detecting ATF2 mRNAlevels.

As described above, the invention provides a screening assay foridentifying proteins that interact with human MEKK. These assays can bedesigned based on the two-hybrid assay system (also referred to as aninteraction trap assay) known in the art (see e.g., Field U.S. Pat. No.5,283,173; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J.Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques14:920-924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696). Thetwo-hybrid assay is generally used for identifying proteins thatinteract with a particular target protein. The assay employs genefusions to identify proteins capable of interacting to reconstitute afunctional transcriptional activator. The transcriptional activatorconsists of a DNA-binding domain and a transcriptional activationdomain, wherein both domains are required to activate transcription ofgenes downstream from a target sequence (such as an upstream activatorsequence (UAS) for GAL4). DNA sequences encoding a target “bait” proteinare fused to either of these domains and a library of DNA sequences isfused to the other domain. “Fish” fusion proteins (generated from thefusion library) capable of binding to the target-fusion protein (e.g., atarget GAL4-fusion “bait”) will generally bring the two domains(DNA-binding domain and transcriptional activation domain) into closeenough proximity to activate the transcription of a reporter geneinserted downstream from the target sequence. Thus, the “fish” proteinscan be identified by their ability to reconstitute a functionaltranscriptional activator (e.g., a functional GAL4 transactivator).

This general two-hybrid system can be applied to the identification ofproteins in cells that interact with human MEKK by construction of atarget human MEKK fusion protein (e.g., a human MEKK/GAL4 binding domainfusion as the “bait”) and a cDNA library of “fish” fusion proteins(e.g., a cDNA/GAL4 activation domain library), wherein the cDNA libraryis prepared from mRNA of a cell type of interest, and introducing theseconstructs into a host cell that also contains a reporter gene constructlinked to a regulatory sequence responsive to human MEKK. cDNAs encodingproteins that interact with human MEKK can be identified based upontransactivation of the reporter gene construct.

Alternatively, a “single-hybrid” assay, such as that described inSieweke, M. H. et al. (1996) Cell 85:49-60, can be used to identifyproteins that interact with human MEKK. This assay is a modification ofthe two-hybrid system discussed above. In this system, the “bait” is atranscription factor from which the transactivation domain has beenremoved (e.g., human MEKK from which the amino-terminal transactivationdomain has been removed) and the “fish” is a non-fusion cDNA library(e.g., a cDNA library prepared from Th2 cells). These constructs areintroduced into host cells (e.g., yeast cells) that also contains areporter gene construct linked to a regulatory sequence responsive tohuman MEKK. cDNAs encoding proteins that interact with human MEKK can beidentified based upon transactivation of the reporter gene construct.

As described above, the invention provides a screening assay foridentifying compounds that modulate the activity of human MEKK byassessing the interaction between ATF 2 and a regulatory element of anATF 2-responsive gene. Assays are known in the art that detect theinteraction of a DNA binding protein with a target DNA sequence (e.g.,electrophoretic mobility shift assays, DNAse I footprinting assays andthe like). By performing such assays in the presence and absence of testcompounds, these assays can be used to identify compounds that modulate(e.g., inhibit or enhance) the interaction of the DNA binding proteinwith its target DNA sequence.

In one embodiment, the amount of binding of ATF 2 to the DNA fragment inthe presence of the test compound is greater than the amount of bindingof ATF 2 to the DNA fragment in the absence of the test compound, inwhich case the test compound is identified as a compound that enhancesactivity of human MEKK. In another embodiment, the amount of binding ofATF 2 to the DNA fragment in the presence of the test compound is lessthan the amount of binding of ATF 2 to the DNA fragment in the absenceof the test compound, in which case the test compound is identified as acompound that inhibits activity of human MEKK.

Yet another aspect of the invention pertains to methods of modulatinghuman MEKK activity in a cell. The modulatory methods of the inventioninvolve contacting the cell with an agent that modulates human MEKKactivity such that human MEKK activity in the cell is modulated. Theagent may act by modulating the activity of human MEKK protein in thecell or by modulating transcription of the human MEKK gene ortranslation of the human MEKK mRNA. As used herein, the term“modulating” is intended to include inhibiting or decreasing human MEKKactivity and stimulating or increasing human MEKK activity. Accordingly,in one embodiment, the agent inhibits human MEKK activity. In anotherembodiment, the agent stimulates human MEKK activity.

C. Inhibitory Agents

According to a modulatory method of the invention, human MEKK activityis inhibited in a cell by contacting the cell with an inhibitory agent.Inhibitory agents of the invention can be, for example, intracellularbinding molecules that act to inhibit the expression or activity ofhuman MEKK. As used herein, the term “intracellular binding molecule” isintended to include molecules that act intracellularly to inhibit theexpression or activity of a protein by binding to the protein itself, toa nucleic acid (e.g., an MRNA molecule) that encodes the protein or to atarget with which the protein indirectly interacts (e.g, to a DNA targetsequence to which ATF 2 binds). Examples of intracellular bindingmolecules, described in further detail below, include antisense humanMEKK nucleic acid molecules (e.g., to inhibit translation of human MEKKmRNA), intracellular anti-human MEKK antibodies (e.g., to inhibit theactivity of human MEKK protein) and dominant negative mutants of thehuman MEKK protein.

In one embodiment, an inhibitory agent of the invention is an antisensenucleic acid molecule that is complementary to a gene encoding humanMEKK or to a portion of said gene, or a recombinant expression vectorencoding said antisense nucleic acid molecule. The use of antisensenucleic acids to downregulate the expression of a particular protein ina cell is well known in the art (see e.g., Weintraub, H. et al,Antisense RNA as a molecular tool for genetic analysis, Reviews—Trendsin Genetics, Vol. 1(1) 1986; Askari, F. K, and McDonnell, W. M. (1996)N. Eng. J. Med. 334:316-318; Bennett, M. R, and Schwartz, S. M. (1995)Circulation 92:1981-1993; Mercola, D. and Cohen, J. S. (1995) CancerGene Ther. 2:47-59; Rossi, J. J. (1995) Br. Med. Bull. 51:217-225;Wagner, R. W. (1994) Nature 372:333-335). An antisense nucleic acidmolecule comprises a nucleotide sequence that is complementary to thecoding strand of another nucleic acid molecule (e.g., an mRNA sequence)and accordingly is capable of hydrogen bonding to the coding strand ofthe other nucleic acid molecule. Antisense sequences complementary to asequence of an mRNA can be complementary to a sequence found in thecoding region of the mRNA, the 5′ or 3′ untranslated region of the mRNAor a region bridging the coding region and an untranslated region (e.g.,at the junction of the 5′ untranslated region and the coding region).Furthermore, an antisense nucleic acid can be complementary in sequenceto a regulatory region of the gene encoding the mRNA, for instance atranscription initiation sequence or regulatory element. Preferably, anantisense nucleic acid is designed so as to be complementary to a regionpreceding or spanning the initiation codon on the coding strand or inthe 3′untranslated region of an mRNA. An antisense nucleic acid forinhibiting the expression of human MEKK protein in a cell can bedesigned based upon the nucleotide sequence encoding the human MEKKprotein (e.g., SEQ ID NO:1), constructed according to the rules ofWatson and Crick base pairing.

An antisense nucleic acid can exist in a variety of different forms. Forexample, the antisense nucleic acid can be an oligonucleotide that iscomplementary to only a portion of a human MEKK gene. An antisenseoligonucleotides can be constructed using chemical synthesis proceduresknown in the art. An antisense oligonucleotide can be chemicallysynthesized using naturally occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleic acids, e.g. phosphorothioatederivatives and acridine substituted nucleotides can be used. To inhibithuman MEKK expression in cells in culture, one or more antisenseoligonucleotides can be added to cells in culture media, typically atabout 200 μg oligonucleotide/ml.

Alternatively, an antisense nucleic acid can be produced biologicallyusing an expression vector into which a nucleic acid has been subclonedin an antisense orientation (i.e., nucleic acid transcribed from theinserted nucleic acid will be of an antisense orientation to a targetnucleic acid of interest). Regulatory sequences operatively linked to anucleic acid cloned in the antisense orientation can be chosen whichdirect the expression of the antisense RNA molecule in a cell ofinterest, for instance promoters and/or enhancers or other regulatorysequences can be chosen which direct constitutive, tissue specific orinducible expression of antisense RNA. For example, for inducibleexpression of antisense RNA, an inducible eukaryotic regulatory system,such as the Tet system (e.g., as described in Gossen, M, and Bujard, H.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995)Science 268:1766-1769; PCT Publication No. WO 94/29442; and PCTPublication No. WO 96/01313) can be used. The antisense expressionvector is prepared as described above for recombinant expressionvectors, except that the cDNA (or portion thereof) is cloned into thevector in the antisense orientation. The antisense expression vector canbe in the form of, for example, a recombinant plasmid, phagemid orattenuated virus. The antisense expression vector is introduced intocells using a standard transfection technique, as described above forrecombinant expression vectors.

In another embodiment, an antisense nucleic acid for use as aninhibitory agent is a ribozvme. Ribozymes are catalytic RNA moleculeswith ribonuclease activity which are capable of cleaving asingle-stranded nucleic acid, such as an mRNA, to which they have acomplementary region (for reviews on ribozymes see e.g., Ohkawa, J. etal. (1995) J. Biochem. 118:251-258; Sigurdsson, S. T, and Eckstein, F.(1995) Trends Biotechnol. 13:286-289; Rossi, J. J. (1995) TrendsBiotechnol. 13:301-306; Kiehntopf, M. et al. (1995) J. Mol. Med.73:65-71). A ribozyme having specificity for human MEKK mRNA can bedesigned based upon the nucleotide sequence of the human MEKK cDNA. Forexample, a derivative of a Tetrahymena L-19 IVS RNA can be constructedin which the base sequence of the active site is complementary to thebase sequence to be cleaved in a human MEKK mRNA. See for example U.S.Pat. Nos. 4,987,071 and 5,116,742, both by Cech et al. Alternatively,human MEKK mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See for exampleBartel, D. and Szostak, J. W. (1993) Science 261: 1411-1418.

Another type of inhibitory agent that can be used to inhibit theexpression and/or activity of human MEKK in a cell is an intracellularantibody specific for the human MEKK protein. The use of intracellularantibodies to inhibit protein function in a cell is known in the art(see e.g., Carlson, J. R. (1988) Mol. Cell. Biol. 8:2638-2646; Biocca,S. et al. (1990) EMBO J. 9:101-108; Werge, T. M. et al. (1990) FEBSLetters 274:193-198; Carlson, J. R. (1993) Proc. Natl. Acad. Sci. USA90:7427-7428; Marasco, W. A. et al. (1993) Proc. Natl. Acad. Sci. USA90:7889-7893; Biocca, S. et al. (1994) Bio/Technology 12:396-399; Chen,S-Y. et al (1994) Human Gene Therapy 5:595-601; Duan, L et al. (1994)Proc. Natl. Acad. Sci. USA 91:5075-5079; Chen, S-Y. et al. (1994) Proc.Natl. Acad Sci. USA 91:5932-5936; Beerli, R. R. et al. (1994) J. Biol.Chem. 269:23931-23936; Beerli, R. R. et al. (1994) Biochem. Biophys.Res. Commun. 204:666-672; Mhashilkar, A. M. et al. (1995) EMBO J.14:1542-1551; Richardson, J. H. et al. (1995) Proc. Natl. Acad. Sci. USA92:3137-3141; PCT Publication No. WO 94/02610 by Marasco et al.; and PCTPublication No. WO 95/03832 by Duan et al.).

To inhibit protein activity using an intracellular antibody, arecombinant expression vector is prepared which encodes the antibodychains in a form such that, upon introduction of the vector into a cell,the antibody chains are expressed as a functional antibody in anintracellular compartment of the cell. For inhibition of human MEKKactivity according to the inhibitory methods of the invention, anintracellular antibody that specifically binds the human MEKK protein isexpressed in the cytoplasm of the cell. To prepare an intracellularantibody expression vector, antibody light and heavy chain cDNAsencoding antibody chains specific for the target protein of interest,e.g., human MEKK, are isolated, typically from a hybridoma that secretesa monoclonal antibody specific for the human MEKK protein. Hybridomassecreting anti-human MEKK monoclonal antibodies, or recombinantanti-human MEKK monoclonal antibodies, can be prepared as describedabove. Once a monoclonal antibody specific for human MEKK protein hasbeen identified (e.g., either a hybridoma-derived monoclonal antibody ora recombinant antibody from a combinatorial library), DNAs encoding thelight and heavy chains of the monoclonal antibody are isolated bystandard molecular biology techniques. For hybridoma derived antibodies,light and heavy chain cDNAs can be obtained, for example, by PCRamplification or cDNA library screening. For recombinant antibodies,such as from a phage display library, CDNA encoding the light and heavychains can be recovered from the display package (e.g., phage) isolatedduring the library screening process. Nucleotide sequences of antibodylight and heavy chain genes from which PCR primers or CDNA libraryprobes can be prepared are known in the art. For example, many suchsequences are disclosed in Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242 and in the“Vbase” human germline sequence database.

Once obtained, the antibody light and heavy chain sequences are clonedinto a recombinant expression vector using standard methods. To allowfor cytoplasmic expression of the light and heavy chains, the nucleotidesequences encoding the hydrophobic leaders of the light and heavy chainsare removed. An intracellular antibody expression vector can encode anintracellular antibody in one of several different forms. For example,in one embodiment, the vector encodes full-length antibody light andheavy chains such that a full-length antibody is expressedintracellularly. in another embodiment, the vector encodes a full-lengthlight chain but only the VH/CH1 region of the heavy chain such that aFab fragment is expressed intracellularly. in the most preferredembodiment, the vector encodes a single chain antibody (scFv) whereinthe variable regions of the light and heavy chains are linked by aflexible peptide linker (e.g., (Gly₄Ser)₃) and expressed as a singlechain molecule. To inhibit human MEKK activity in a cell, the expressionvector encoding the anti-human MEKK intracellular antibody is introducedinto the cell by standard transfection methods, as discussedhereinbefore.

Yet another form of an inhibitory agent of the invention is aninhibitory form of human MEKK, also referred to herein as a dominantnegative inhibitor. The MEKK proteins are known to modulate the activityof MEKK target molecules, particularly by modulating the phosphorylationstate of the MEKK target molecule. One means to inhibit the activity ofmolecule that has an enzymatic activity is through the use of a dominantnegative inhibitor that has the ability to interact with the targetmolecule but that lacks enzymatic activity. By interacting with thetarget molecule, such dominant negative inhibitors can inhibit theactivation of the target molecule. This process may occur naturally as ameans to regulate enzymatic activity of a cellular signal transductionmolecule.

Accordingly, an inhibitory agent of the invention can be a form of ahuman MEKK protein that has the ability to interact with other proteinsbut that lacks enzymatic activity. This dominant negative form of ahuman MEKK protein may be, for example, a mutated form of human MEKK inwhich a kinase consensus sequence has been altered. Such dominantnegative human MEKK proteins can be expressed in cells using arecombinant expression vector encoding the human MEKK protein, which isintroduced into the cell by standard transfection methods. The mutatedDNA is inserted into a recombinant expression vector, which is thenintroduced into a cell to allow for expression of the mutated humanMEKK, lacking enzymatic activity.

Other inhibitory agents that can be used to inhibit the activity of ahuman MEKK protein are chemical compounds that directly inhibit humanMEKK activity or inhibit the interaction between human MEKK and targetmolecules. Such compounds can be identified using screening assays thatselect for such compounds, as described in detail above.

D. Stimulatory Agents

According to a modulatory method of the invention, human MEKK activityis stimulated in a cell by contacting the cell with a stimulatory agent.Examples of such stimulatory agents include active human MEKK proteinand nucleic acid molecules encoding human MEKK that are introduced intothe cell to increase human MEKK activity in the cell. A preferredstimulatory agent is a nucleic acid molecule encoding a human MEKKprotein, wherein the nucleic acid molecule is introduced into the cellin a form suitable for expression of the active human MEKK protein inthe cell. To express a human MEKK protein in a cell, typically a humanMEKK-encoding DNA is first introduced into a recombinant expressionvector using standard molecular biology techniques, as described herein.A human MEKK-encoding DNA can be obtained, for example, by amplificationusing the polymerase chain reaction (PCR), using primers based on thehuman MEKK nucleotide sequence. Following isolation or amplification ofhuman MEKK-encoding DNA, the DNA fragment is introduced into anexpression vector and transfected into target cells by standard methods,as described herein.

Other stimulatory agents that can be used to stimulate the activity of ahuman MEKK protein are chemical compounds that stimulate human MEKKactivity in cells, such as compounds that directly stimulate human MEKKprotein and compounds that promote the interaction between human MEKKand target molecules. Such compounds can be identified using screeningassays that select for such compounds, as described in detail above.

The modulatory methods of the invention can be performed in vitro (e.g.,by culturing the cell with the agent or by introducing the agent intocells in culture) or, alternatively, in vivo (e.g., by administering theagent to a subject or by introducing the agent into cells of a subject,such as by gene therapy). For practicing the modulatory method in vitro,cells can be obtained from a subject by standard methods and incubated(i.e., cultured) in vitro with a modulatory agent of the invention tomodulate human MEKK activity in the cells. For example, peripheral bloodmononuclear cells (PBMCs) can be obtained from a subject and isolated bydensity gradient centrifugation, e.g., with Ficoll/Hypaque. Specificcell populations can be depleted or enriched using standard methods. Forexample, monocytes/macrophages can be isolated by adherence on plastic.B cells can be enriched for example, by positive selection usingantibodies to B cell surface markers, for example by incubating cellswith a specific primary monoclonal antibody (mAb), followed by isolationof cells that bind the mAb using magnetic beads coated with a secondaryantibody that binds the primary mAb. Specific cell populations can alsobe isolated by fluorescence activated cell sorting according to standardmethods. If desired, cells treated in vitro with a modulatory agent ofthe invention can be readministered to the subject. For administrationto a subject, it may be preferable to first remove residual agents inthe culture from the cells before administering them to the subject.This can be done for example by a Ficoll/Hypaque gradient centrifugationof the cells. For further discussion of ex vivo genetic modification ofcells followed by readministration to a subject, see also U.S. Pat. No.5,399,346 by W. F. Anderson et al.

For practicing the modulatory method in vivo in a subject, themodulatory agent can be administered to the subject such that human MEKKactivity in cells of the subject is modulated. The term “subject” isintended to include living organisms in which a MEKK-dependent cellularresponse can be elicited. Preferred subjects are mammals. Examples ofsubjects include humans, monkeys, dogs, cats, mice, rats, cows, horses,goats and sheep. For stimulatory or inhibitory agents that comprisenucleic acids (including recombinant expression vectors encoding humanMEKK protein, antisense RNA, intracellular antibodies or dominantnegative inhibitors), the agents can be introduced into cells of thesubject using methods known in the art for introducing nucleic acid(e.g., DNA) into cells in vivo. Examples of such methods encompass bothnon-viral and 20. viral methods, including:

Direct Injection: Naked DNA can be introduced into cells in vivo bydirectly injecting the DNA into the cells (see e.g., Acsadi et al.(1991) Nature 332:815-818; Wolff et al. (1990) Science 247:1465-1468).For example, a delivery apparatus (e.g., a “gene gun”) for injecting DNAinto cells in vivo can be used. Such an apparatus is commerciallyavailable (e.g., from BioRad).

Cationic Lipids: Naked DNA can be introduced into cells in vivo bycomplexing the DNA with cationic lipids or encapsulating the DNA incationic liposomes. Examples of suitable cationic lipid formulationsinclude N-[-1-(2,3-dioleoyloxy)propyl]N,N,N-triethylammonium chloride(DOTMA) and a 1:1 molar ratio of1,2-dimyristyloxy-propyl-3-dimethylhydroxyethylammonium bromide (DMRIE)and dioleoyl phosphatidylethanolamine (DOPE) (see e.g., Logan, J. J. etal. (1995) Gene Therapy 2:38-49; San, H. et al. (1993) Human GeneTherapy 4:781-788).

Receptor-Mediated DNA Uptake: Naked DNA can also be introduced intocells in vivo by complexing the DNA to a cation, such as polylysine,which is coupled to a ligand for a cell-surface receptor (see forexample Wu, G, and Wu, C. H. (1988) J. Biol. Chem. 263:14621; Wilson etal. (1992) J. Biol. Chem. 267:963-967; and U.S. Pat. No. 5,166,320).Binding of the DNA-ligand complex to the receptor facilitates uptake ofthe DNA by receptor-mediated endocytosis. A DNA-ligand complex linked toadenovirus capsids which naturally disrupt endosomes, thereby releasingmaterial into the cytoplasm can be used to avoid degradation of thecomplex by intracellular lysosomes (see for example Curiel et al. (1991)Proc. Nati. Acad. Sci USA 88:8850; Cristiano et al. (1993) Proc. Natl.Acad. Sci. USA 90:2122-2126).

Retroviruses: Defective retroviruses are well characterized for use ingene transfer for gene therapy purposes (for a review see Miller, A. D.(1990) Blood 76:271). A recombinant retrovirus can be constructed havinga nucleotide sequences of interest incorporated into the retroviralgenome. Additionally, portions of the retroviral genome can be removedto render the retrovirus replication defective. The replicationdefective retrovirus is then packaged into virions which can be used toinfect a target cell through the use of a helper virus by standardtechniques. Protocols for producing recombinant retroviruses and forinfecting cells in vitro or in vivo with such viruses can be found inCurrent Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.)Greene Publishing Associates, (1989), Sections 9.10-9.14 and otherstandard laboratory manuals.

Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM whichare well known to those skilled in the art. Examples of suitablepackaging virus lines include ψCrip, ψCre, ψ2 and ψAm. Retroviruses havebeen used to introduce a variety of genes into many different celltypes, including epithelial cells, endothelial cells, lymphocytes,myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (seefor example Eglitis, et al. (1985) Science 230:1395-1398; Danos andMulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al.(1988) Proc. Natl. Acad. Sci USA 85:3014-3018; Armentano et al. (1990)Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al. (1991) Proc. Natl.Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci.USA 88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; vanBeusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay etal. (1992) Human Gene Therapy 3:641-647; Dai et al. (1992) Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et al. (1993) J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Retroviral vectors requiretarget cell division in order for the retroviral genome (and foreignnucleic acid inserted into it) to be integrated into the host genome tostably introduce nucleic acid into the cell. Thus, it may be necessaryto stimulate replication of the target cell.

Adenoviruses: The genome of an adenovirus can be manipulated such thatit encodes and expresses a gene product of interest but is inactivatedin terms of its ability to replicate in a normal lytic viral life cycle.See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld etal. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell68:143-155. Suitable adenoviral vectors derived from the adenovirusstrain Ad type 5 d1324 or other strains of adenovirus (e.g., Ad2, Ad3,Ad7 etc.) are well known to those skilled in the art. Recombinantadenoviruses are advantageous in that they do not require dividing cellsto be effective gene delivery vehicles and can be used to infect a widevariety of cell types, including airway epithelium (Rosenfeld et al.(1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc.Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993)Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin etal. (1992) Proc. Nati. Acad. Sci. USA 89:2581-2584). Additionally,introduced adenoviral DNA (and foreign DNA contained therein) is notintegrated into the genome of a host cell but remains episomal, therebyavoiding potential problems that can occur as a result of insertionalmutagenesis in situations where introduced DNA becomes integrated intothe host genome (e.g., retroviral DNA). Moreover, the carrying capacityof the adenoviral genome for foreign DNA is large (up to 8 kilobases)relative to other gene delivery vectors (Berkner et al. cited supra;Haj-Ahmand and Graham (1986) J. Virol. 57:267). Mostreplication-defective adenoviral vectors currently in use are deletedfor all or parts of the viral E1 and E3 genes but retain as much as 80%of the adenoviral genetic material.

Adeno-Associated Viruses: Adeno-associated virus (AAV) is a naturallyoccurring defective virus that requires another virus, such as anadenovirus or a herpes virus, as a helper virus for efficientreplication and a productive life cycle. (For a review see Muzyczka etal. Curr. Topics in Micro, and Immunol. (1992) 158:97-129). It is alsoone of the few viruses that may integrate its DNA into non-dividingcells, and exhibits a high frequency of stable integration (see forexample Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol. 7:349-356;Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al.(1989) J. Virol. 62:1963-1973). Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate. Space for exogenous DNAis limited to about 4.5 kb. An AAV vector such as that described inTratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used tointroduce DNA into cells. A variety of nucleic acids have beenintroduced into different cell types using AAV vectors (see for exampleHermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470;Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al(1988) Mol. Endocrinol. 2:32-39; Tratschin et al. (1984) J. Virol.51:611-619; and Flotte et al. (1993) J. Biol. Chem. 268:3781-3790).

The efficacy of a particular expression vector system and method ofintroducing nucleic acid into a cell can be assessed by standardapproaches routinely used in the art. For example, DNA introduced into acell can be detected by a filter hybridization technique (e.g., Southernblotting) and RNA produced by transcription of introduced DNA can bedetected, for example, by Northern blotting, RNase protection or reversetranscriptase-polymerase chain reaction (RT-PCR). The gene product canbe detected by an appropriate assay, for example by immunologicaldetection of a produced protein, such as with a specific antibody, or bya functional assay to detect a functional activity of the gene product.

In a preferred embodiment, a retroviral expression vector encoding humanMEKK is used to express human MEKK protein in cells in vivo, to therebystimulate MEKK protein activity in vivo. Such retroviral vectors can beprepared according to standard methods known in the art (discussedfurther above).

A modulatory agent, such as a chemical compound, can be administered toa subject as a pharmaceutical composition. Such compositions typicallycomprise the modulatory agent and a pharmaceutically acceptable carrier.As used herein the term “pharmaceutically acceptable carrier” isintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active compound, use thereof in thecompositions is contemplated. Supplementary active compounds can also beincorporated into the compositions. Pharmaceutical compositions can beprepared as described above in subsection IV.

This invention is further illustrated by the following example, whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are hereby incorporated by reference.

Additionally, all nucleotide and amino acid sequences deposited inpublic databases referred to herein are also hereby incorporated byreference.

EXAMPLE 1

Isolation and Characterization of a Human MEKK1 Nucleic Acid

The following strategy was used to identify the nucleic acid sequenceencoding human MEKK1.

cDNA Preparation—Total MRNA was extracted and isolated from T47D cellsusing 1×10⁷ cells per purification in the QuickPrep Micro mRNAPurification Kit (Pharmacia). First strand cDNA was produced using 33microliters of the purified mRNA per reaction in the Ready-to-GoT-Primed First-Strand Kit (Pharmacia).

PCR Amplification—The sense strand primer 5′-GAACACCATCCAGAAGTTTG-3′(SEQ ID NO:14), which was designed from the mouse MEKK1 (mMEKK1) cDNAsequence, was used in conjunction with the antisense primer5′-CACTTTGTAGACAGGGTCAGC-3′ (SEQ ID NO:15) in a polymerase chainreaction (PCR) using the first strand cDNA described above as a template(RT-PCR) to amplify the region from bases 1211-1950. Taq DNA Polymerase(Boehringer Mannheim) was used in a RT-PCR of 30 cycles (1 min. 94° C.;1 min. 50° C.; 3 min., 72° C.), followed by a 10 min. incubation at 72°C. A band of approximately 800 bp was isolated by purification from a 1%agarose gel and ligated overnight at 14° C. into pGEM-T coli by heatshock at 42° C., and plated on Luria Broth (LB) plates containingampicillin and X-gal. Colonies were screened by blue/white colorselection, grown up in 5 ml of LB containing ampicillin, and the plasmidDNA was isolated using the Wizard Mini-pre Kit (Promega). Isolates werethen screened for insert size by digesting with PstI and AatII(Promega), and running on a 1% agarose gel. Appropriately sized insertswere sequenced from both ends using T7 and SP6 vector primers. Theresulting sequence was aligned to the known mMEKK1 sequence, anddetermined to be hMEKK1 by homology. In order to amplify the region frombases 2263-3743, the sense primer 5′-TGGGTCGCCTCTGTCTTATAGACAG-3′ (SEQID NO:16) was used in conjunction with the antisense primer5′-CACATCCTGTGCTTGGTAAC-3′ (SEQ ID NO:17) in a RT-PCR of 30 cycles (1min. 94° C.; 1 min., 50° C.; 2 min., 72° C.), followed by a 10 min.incubation at 72° C. A band of approximately 1.5 kb was isolated bypurification from a 1% agarose gel, ligated, cloned, and sequenced asstated above. In order to amplify the 3′ region of hMEKK1 from bases3304-4493, the sense primer 5′-AGGACAAGTGCAGGTTAGATG-3′ (SEQ ID NO:18)was used in a RT-PCR of 30 cycles (1 min., 94° C.; 1 min., 50° C.; 2min., 72° C.), followed by a 10 min. incubation at 72° C. A band ofapproximately 1.3 kb was isolated by purification from a 1% agarose gel,ligated, cloned, and sequenced as stated above. Sequence was alsoconfirmed for this clone using the internal sequencing primer5′-GCTGTCCATATCTACAGTGCT-3′ (SEQ ID NO:19). In order to amplify theregion from bases 580-1310, the sense primer 5′-CGGCCTGGAAGCACGAGTGGT-3′(SEQ ID NO:20) was used in conjunction with the antisense primer5′-TTCATCCTTGATGCTGTTTTC-3′ (SEQ ID NO:21) in a RT-PCR of 30 cycles (1min., 94° C.; 1 min., 50° C.; 2 min., 72° C.), followed by a 10 min Aband of approximately 700 bp was isolated by purification from a 1%agarose gel, ligated, cloned, and sequenced as stated above. Theoverlapping sequence data was compiled into a single contig usingSequencer 2.0 (Gene Codes), and aligned to the mMEKK1 sequence (FIG. 3).The nucleotide and predicted amino acid sequences of human MEKK3 areshown in FIGS. 1 and 2, respectively. FIG. 4 depicts an alignment of theamino acid sequences of hMEKK1 and mMEKK1. Amino acid differencesbetween the two proteins are indicated by bold underlining.

A BLASTN search using the nucleotide sequence of human MEKK1 asdescribed in this example reveals the following nucleic acid sequenceshaving homology to that set forth in SEQ ID NO:1: GenBank Accession Nos.L13103, U23470, AF042838, and U48596, having 96%, 96%, 95%, and 89%identity to SEQ ID NO:1, respectively.

A BLASTP search using the amino acid sequence of human MEKK1 asdescribed in this example reveals the following amino acid sequenceshaving homology to that set forth in SEQ ID NO:2: Swiss-Prot AccessionNo. Q62925, having 82% identity to SEQ ID NO :2; GenBank Accession No.AF042838, having 81% identity to SEQ ID NO:2; and GenBank Accession No.P53349, GenBank Accession No. U23470, PIR Accession No. A46212, andGenBank Accession No. L13103, each having 91% identity to SEQ ID NO:2;having 91% identity to SEQ ID NO:2.

EXAMPLE 2

Isolation and Characterization of a Human MEKK2 Nucleic Acid

The following strategy was used to identify the nucleic acid sequenceencoding human MEKK2.

CDNA Preparation—Total mRNA was extracted and isolated from T47D cellsusing 1×10⁷ cells per purification in the QuickPrep Micro mRNAPurification Kit (Pharmacia). First strand cDNA was produced using 33microlitres of the purified mRNA per reaction in the Ready-To-GoT-Primed First-Strand Kit (Pharmacia).

PCR Amplification—The sense strand primer 5′-GGCCAGCTCGGTGGCCT-3′ (SEQID NO:22), which annealed to the 5′ untranslated region of human MEKK2(hMEKK2), was designed from the mouse MEKK2 (mMEKK2) cDNA sequence, andused in conjunction with the antisense primer 5′-TCTGGAATGTATCCTGG-3′(SEQ ID NO:23) in a polymerase chain reaction (PCR) using the firststrand cDNA described above as a template (RT-PCR). Taq DNA Polymerase(Boehringer Mannheim) was used in a RT-PCR of 30 cycles (1 min, 94° C.;1 min, 50° C.; 3 min, 68° C.), followed by a 10-min incubation at 72° C.One microlitre of the resulting reaction mixture was used as a templatefor a second PCR under the same conditions, and one microlitre of thesecondary reaction mixture was used as a template again in a third PCRunder the same conditions. A band of approximately 600 bp was isolatedby purification from a 1% agarose gel and ligated overnight at 14° C.into pCR2.1 using the Original T/A Cloning Kit (Invitrogen). Theligation mixture was transformed by heat shock of the E. Coli strainTOP10F′ at 42° C., and plated on Luria Broth (LB) plates containingampicillin and X-gal. Colonies were screened by blue/white colorselection, grown up in 5 ml of LB containing ampicillin, and the plasmidDNA was isolated using Mini-Prep Spin Columns (Qiagen). Isolates werethen screened for insert size by digesting with EcoRI (Gibco/BRL), andrunning on a 1% agarose gel. Appropriately sized inserts were sequencedfrom both ends using M13 Forward and Reverse vector primers. Theresulting sequence was aligned to the known mMEKK2 sequence, anddetermined to be hMEKK2 by homology. After sequencing the 5′ portion ofhMEKK2, the sense primer 5′-AGAGAGGAAAAAGCGGC-3′ (SEQ ID NO:24), whichannealed to a region that overlapped the previously sequenced portion ofhMEKK2, was used in conjunction with the two antisense primers5′-CAGCCAGCTCTCTTCCG-3′ (SEQ ID NO:25) and 5′-GGAAAAGTCTTCCGACC-3′ (SEQID NO:26) in two separate RT-PCR of 30 cycles (1 min, 94° C.; 1 min, 50°C.; 2 min, 68° C.), followed by a 10-min incubation at 72° C. Onemicrolitre of the resulting reaction mixtures was used as a template fortwo separate second PCR under the same conditions. Two bands ofapproximately 700 bp and 400 bp respectively were isolated bypurification from a 1% agarose gel, ligated, cloned, and sequenced asstated above. In order to sequence the 3′ portion of hMEKK2, the senseprimer 5′-GGCCAAGGAGCTTTTGGTAGG-3′ (SEQ ID NO:27), which annealed to aregion that overlapped the previously sequenced region of hMEKK2, wasused in conjunction with the antisense primer5′-GGAGCTGGTGGAGGACCGAAG-3′ (SEQ ID NO:28), which annealed to the 3′untranslated region of hMEKK2, in a RT-PCR of 30 cycles (1 min, 94° C.;1 min, 50° C.; 2 min, 68° C.), followed by a 10-min incubation at 72° C.One microlitre of the resulting reaction mixture was used as a templatefor a second PCR under the same conditions. A band of approximately 750bp was isolated by purification from a 1% agarose gel, ligated, cloned,and sequenced as stated above. The overlapping sequence data wascompiled into a single contig using Sequencher 2.0 (Gene Codes), andaligned to the mMEKK2 sequence (FIG. 7). The nucleotide and predictedamino acid sequences of human MEKK3 are shown in FIGS. 5 and 6,respectively. FIG. 8 depicts an alignment of the amino acid sequences ofhMEKK2 and mMEKK2. Amino acid differences between the two proteins areindicated by bold underlining.

A BLASTN search using the nucleotide sequence of human MEKK2 asdescribed in this example reveals that a nucleic acid molecule havingGenBank Accession No. U43186 has 98% identity to the human MEKK2 nucleicacid sequence set forth as SEQ ID NO:3. A BLASTP search using the humanMEKK2 amino acid sequence set forth in SEQ ID NO:4 reveals the followingproteins having homology to human MEKK2: Swiss Prot Accession Nos.Q61083, Q61084, and Q99759 having 90%, 63% and 63% identity,respectively.

EXAMPLE 3

Isolation and Characterization of a Human MEKK3 Nucleic Acid

To isolate a nucleic acid molecule encoding human MEKK3, the senseprimer 5′-CCCAGAACCCTGGCCGAAGCT-3′ (SEQ ID NO:29), which annealed to aregion in the middle of human MEKK3 (hMEKK3), was designed from themouse MEKK3 (mMEKK3) cDNA sequence and used in conjunction with theantisense primer 5′-AGCACGGTCCCGCAGGCAGCC-3) (SEQ ID NO:30). Taq DNAPolymerase (Boehringer Mannheim) was used in a RT-PCR of 30 cycles (1min, 94° C.; 1 min, 50° C.; 3 min, 72° C.), followed by a 10-minincubation at 72° C. using Marathon ReadyT™ human bone marrow,placental, and testis cDNA (Clontech) as templates. A band ofapproximately 800 bp was isolated from testis and placental templatereactions by purification from a 1% agarose gel and ligated overnight at14° C. into pCR2.1 using the Original T/A Cloning Kit (Invitrogen). Theligation mixture was transformed by heat shock of the E. coli strainTOP10F′ at 42° C., and plated on Luria Broth (LB) plates containingampicillin, IPTG and X-gal. Colonies were screened by blue/white colorselection, grown up in 5ml of LB containing ampicillin, and the plasmidDNA was isolated using Mini-Prep™ Spin Columns (Qiagen). Isolates werethen screened for insert size by digesting with EcoRI (Gibco/BRL), andrunning on a 1% agarose gel. Appropriately sized inserts were sequencedfrom both ends using M13 Forward and Reverse vector primers. Theresulting sequence was aligned to the known mMEKK3 sequence, anddetermined to be hMEKK3 by homology. In order to sequence the 5′ end ofHMEKK, the sense strand primer 5′-GTAGTCGCCACCGCCGCCTCC-3′ (SEQ ID NO:31), which annealed to the 5′ untranslated region of hMEKK3, wasdesigned from the mMEKK3 cDNA sequence, and used in conjunction with theantisense primer 5′-CTGACAAGGAATTTTCGGCAC-3′ (SEQ ID NO:32) whichoverlapped the previously sequenced portion of hMEKK3, in a RT-PCR of 30cycles (1 min, 94° C.; 1 min, 50° C.; 3 min, 72° C.) in seven differentbuffers of varying pH and magnesium concentrations, followed by a 10-minincubation at 72° C. One microlitre of the resulting reaction mixturewere used as a template for a second PCR under the same conditions withthe nested sense strand oligo 5′-ACCGCCGCCTCCGCCATCGCC-3′ (SEQ ID NO:33)and the nested antisense strand oligo 5′-CACTGTTCGCTGGTCTCTGGG-3′ (SEQID NO:34). A band of approximately 700 bp was isolated from the reactionmixture buffered with 1 7.5mM MgCl₂ at pH 8.5 by purification from a 1%agarose gel, ligated, cloned, and sequenced as stated above. In order tosequence the 3′ portion of hMEKK3, the sense primer5′-AGACAAGCAAGGAGGTGAGTG-3′ (SEQ ID NO:35), which annealed to a regionthat overlapped the previously sequenced region of hMEKK3, was used inconjunction with the antisense primer 5′-GCCTGACAGCAGCCCCTTGCC-3′ (SEQID NO:36), which annealed to the 3′ untranslated region of hMEKK3, in aRT-PCR of 30 cycles (1 min, 94° C.; 1 min, 50° C.; 2 min, 72° C.),followed by a 10-min incubation at 72° C. Subsequently, the nested senseprimer 5′-TCCAGTTGCTAAAGAACTTGC-3′ (SEQ ID NO:37) was used inconjunction with the nested antisense primer 5′-TGGCAGCTGGCAGCCTGATAG-3′(SEQ ID NO:38) in a secondary RT-PCR of 30 cycles (1 min, 94° C.; 1 min,50° C.; 2 min, 72° C.), followed by a 10-min incubation at 72° C. A bandof approximately 670 bp was isolated by purification from a 1% agarosegel, ligated, cloned, and sequenced as stated above. The overlappingsequence data was compiled into a single contig using Sequencher 2.0(Gene Codes), and aligned to the mMEKK3 sequence (FIG. 11). Thenucleotide and predicted amino acid sequences of human MEKK3 are shownin FIGS. 9 and 10, respectively. FIG. 12 depicts an alignment of theamino acid sequences of hMEKK3 and mMEKK3. Amino acid differencesbetween the two proteins are evident, which differences are underlinedand bolded.

A BLASTN search using the nucleic acid sequence of human MEKK3 revealsthe following nucleic acid molecules having homology to SEQ ID NO:5:GenBank Accession Nos. U43187 and U78876, having 95% and 93% identity,respectively. A BLASTP search using the human MEKK3 amino acid sequenceset forth in SEQ ID NO:6 reveals the following proteins having homologyto human MEKK3: Swiss Prot Accession Nos. Q61084, Q99759, and Q61083,having 97%, 95%, and 62% identity to SEQ ID NO:6.

EXAMPLE 4

Antibodies to Human MEKK Proteins

Peptides corresponding to COOH-terninal sequence of a human MEKK protein(e.g., amino acids 1280-1300 of SEQ ID NO:2, amino acids 599-617 of SEQID NO:4, or amino acids 605-623 of SEQ ID NO:6) are conjugated tokeyhole limpet hemocyanin and used to irnmunize rabbits. Antisera arecharacterized for specificity by immunoblotting of lysates prepared fromappropriately transfected HEK293 cells.

EXAMPLE 5

Assays of Activity of MEKK and Downstream Signaling Molecules

Assay ofJNK Activity—JNK activity is measured using GST (glutathioneS-transferase)-c-Jun₍₁₋₇₉₎ coupled to glutathione-Sepharose 4B (M. Hibiet al., Genes & Dev.; 7:2135-2148 (1993)). Cells transfected with MEEK3and control transfected cells are lysed in 0.5% Nonidet P-40, 20 mMTris-HCL, pH 7.6, 0.25 M NaCI, 3 mM EDTA, 3 mM EGTA, 1 mMdithiothreitol, 1 mM phenymethylsulfonyl fluoride, 2 mM sodium vanadate,20 μ/ml aprotinin, and 5 μg/ml leupeptin. Nuclei are removed bycentrifugation at 15,000×g for 10 min., and the supernatants (25 μg ofprotein) are mixed with 10 μl of slurry of GST-c-Jun₍₁₋₇₉₎-Sepharose(3-5 μg of GST-c-Jun₍₁₋₇₉₎. The mixture is rotated at 4° C. for 1 h,washed trice in lysis buffer and once in kinase buffer (20 mM Hepes, pH7.5, 10 mM MgCl₂, 20 mM β-glycerophosphate, 10 mM p-nitrophenylphosphate, 1 mM dithiothreitol, 50 μM sodium vanadate). Beads aresuspended in 40 μl of kinase assay buffer containing 10 μCi of[γ-³²P]ATP and incubated at 30° C. for 20 minutes. Reactions mixturesare added to Laemmli sample buffer, boiled, and phosphorylated proteinsare resolved on SDS-01% polyacrylamide gels. When JNK activity isassayed following fractionation by Mono Q ion exchange chromatography,50 μl of each fraction is incubated with the GST-c-Jun₍₁₋₇₉₎ beads.

p42/44^(MAPK) Assay—MAPK activity following Mono Q PPLC fractionation ismeasured as described in L. E. Heasley et al., Mol. Biol. Cell;3:545-533 (1992) using the epidermal growth factor receptor 662-681peptide as a selective p42/44^(MAPK) substrate (M. Russell et al.,Biochemistry; 34:6611-6615 (1995)). Alternatively, for cells transfectedwith varying amounts of MEKK plasmids MAPK activity is assayed afterelution from DEAE-Sephacel columns (L. E. Heasley et al., Am. J.Physiol.; 267:F366-F373 (1994)).

Assay ofMEKK Kinase Activity in Vitro—To assay MEKK activity in vitro,immune complexes are incubated with recombinant wild type orkinase-inactive MEK 1 (Lys⁹⁷→Met) or JNKK (Lys¹¹⁶→Arg) as a substrate(A. Lin et al, Science; 268:286-290 (1995), C. A. Lange-Carter et al,Science; 265:1458-1461 (1994), M. Russell et al., Biochemistry;34:6611-6615 (1995)). Transfected HEK293 cells are lysed in 1% TritonX-100, 0.5% Nonidet P-40, 20 mM Tris-HCl, pH 7.5, 150 mM NaCL, 20 mMNaF, 0.2 mM sodium vanadate, 1 mM EDTA, 1 mM EGTA, 5 mMphenylmethylsulfonyl fluoride. Nuclei are removed by centrifugation at15,000×g for 5 min. HA epitope-tagged MEKK are immunoprecipitated withthe 12CA5 antibody and protein A-Sepharose (B. E. Wadzinski et al., J.Biol. Chem.; 267:16883-16888 (1992), N.-X. Qian et al., Proc. Natl.Acad. Sci. U.S.A.; 90:40774081 (1993)). Immunoprecipitates are washedtwice in lysis buffer, twice in 20 mM Pipes, 10 mM MnCl₂, 20 μg/mlaprotinin, and used in an in vitro kinase assay with 20-50 ng ofrecombinant MEK 1 or JNKK as substrates and 20 μCi of [γ-³²P]ATP (M.Russell et al., Biochemistry; 34:6611-6615 (1995)). Reactions areterminated by the addition of Laemmli sample buffer, boiled, andproteins are resolved by SDS-10% polyacrylamide gel electrophoresis.

To demonstrate MEKK activation of JNKK activity, the in vitro kinasereactions are performed with different combinations of recombinant wildtype or kinase-inactive JNK. Kinase-inactive NJK is made by mutating theactive site lysine 55 to methionine. Incubations are for 30 min, at 30°C. in the presence of 50 μM ATP. GST-c-Jun₍₁₋₇₉₎-Sepharose beads arethen added, and the mixture is rotated at 4° C. for 30 minutes. Thebeads are washed, suspended in 40 μl of c-Jun kinase assay buffercontaining 20 μCi of [γ-³²P]ATP, and incubated for 15 min, at 30° C.Reaction mixtures are added to Laemmli sample buffer, boiled, andphosphorylated proteins are resolved in SDS-10% polyacrylamide gels.

Assay ofp38 Kinase Activity—Sorbitol-treated (0.4 M, 20 min.) or controlHEK293 cells are lysed in the same buffer as that used for assay ofMEKK. Supernatants (200 μg of protein) are used for COOH-terminalpeptide sequence of p38 (J. Han et al, Science; 265:8-8-811 (1994)).Immunoprecipitates are washed once in lysis buffer, once in assay buffer(25 mM Hepes, pH 7.4, sodium vanadate), resuspended, and used in an invitro kinase assay susbstrate and 20 μCi of [γ-³²P]ATP (H. Abdel-Hafezet al., Mol. Endocrinology; 6:2079-2089 (1992)). For analysis of p38kinase activity from Mono Q FPLC fractions, 20 μl aliquots are mixedwith kinase buffer containing 20-50 ng of recombinant ATF 2 and 10 μCiof [γ-³²P]ATP (M. Russell et al., Biochemistry; 34:6611-6615 (1995), H.Abdel-Hafez et al., Mol. Endocrinology; 6:2079-2089 (1992)). Reactionsare quenched in Laemmli sample buffer, boiled, and proteins are resolvedusing SDS-10% polyacrylamide gels.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

38 1 3911 DNA Homo sapiens CDS (3)..(3908) 1 cg gcc tgg aag cac gag tggttg gaa agg aga aat agg cga ggg cct 47 Ala Trp Lys His Glu Trp Leu GluArg Arg Asn Arg Arg Gly Pro 1 5 10 15 gtg gtg gta aaa cca atc cca gttaaa gga gat gga tct gaa atg aat 95 Val Val Val Lys Pro Ile Pro Val LysGly Asp Gly Ser Glu Met Asn 20 25 30 cac tta gca gct gag tct cca gga gaggtc cag gca agt gcg gct tca 143 His Leu Ala Ala Glu Ser Pro Gly Glu ValGln Ala Ser Ala Ala Ser 35 40 45 cca gct tcc aaa ggc cga cgc agt cct tctcct ggc aac tcc cca tca 191 Pro Ala Ser Lys Gly Arg Arg Ser Pro Ser ProGly Asn Ser Pro Ser 50 55 60 ggt cgc aca gtg aaa tca gaa tct cca gga gtaagg aga aaa aga gtt 239 Gly Arg Thr Val Lys Ser Glu Ser Pro Gly Val ArgArg Lys Arg Val 65 70 75 tcc cca gtg cct ttt cag agt ggc aga atc aca ccaccc cga aga gcc 287 Ser Pro Val Pro Phe Gln Ser Gly Arg Ile Thr Pro ProArg Arg Ala 80 85 90 95 cct tca cca gat ggc ttc tca cca tat agc cct gaggaa aca aac cgc 335 Pro Ser Pro Asp Gly Phe Ser Pro Tyr Ser Pro Glu GluThr Asn Arg 100 105 110 cgt gtt aac aaa gtg atg cgg gcc aga ctg tac ttactg cag cag ata 383 Arg Val Asn Lys Val Met Arg Ala Arg Leu Tyr Leu LeuGln Gln Ile 115 120 125 ggg cct aac tct ttc ctg att gga gga gac agc ccagac aat aaa tac 431 Gly Pro Asn Ser Phe Leu Ile Gly Gly Asp Ser Pro AspAsn Lys Tyr 130 135 140 cgg gtg ttt att ggg cct cag aac tgc agc tgt gcacgt gga aca ttc 479 Arg Val Phe Ile Gly Pro Gln Asn Cys Ser Cys Ala ArgGly Thr Phe 145 150 155 tgt att cat ctg cta ttt gtg atg ctc cgg gtg tttcaa cta gaa cct 527 Cys Ile His Leu Leu Phe Val Met Leu Arg Val Phe GlnLeu Glu Pro 160 165 170 175 tca gac cca atg tta tgg aga aaa act tta aagaat ttt gag gtt gag 575 Ser Asp Pro Met Leu Trp Arg Lys Thr Leu Lys AsnPhe Glu Val Glu 180 185 190 agt ttg ttc cag aaa tat cac agt agg cgt agctca agg atc aaa gct 623 Ser Leu Phe Gln Lys Tyr His Ser Arg Arg Ser SerArg Ile Lys Ala 195 200 205 cca tct cgt aac acc atc cag aag ttt gtt tcacgc atg tca aat tct 671 Pro Ser Arg Asn Thr Ile Gln Lys Phe Val Ser ArgMet Ser Asn Ser 210 215 220 cat aca ttg tca tca tct agt act tct aca tctagt tca gta aac agc 719 His Thr Leu Ser Ser Ser Ser Thr Ser Thr Ser SerSer Val Asn Ser 225 230 235 ata aag gat gaa gag gaa cag atg tgt cct atttgc ttg ttg ggc atg 767 Ile Lys Asp Glu Glu Glu Gln Met Cys Pro Ile CysLeu Leu Gly Met 240 245 250 255 ctt gat gaa gaa agt ctt aca gtg tgt gaagac ggc tgc agg aac aag 815 Leu Asp Glu Glu Ser Leu Thr Val Cys Glu AspGly Cys Arg Asn Lys 260 265 270 ctg cac cac cac tgc atg tca att tgg gcagaa gag tgt aga aga aat 863 Leu His His His Cys Met Ser Ile Trp Ala GluGlu Cys Arg Arg Asn 275 280 285 aga gaa cct tta ata tgt ccc ctt tgt agatct aag tgg aga tct cat 911 Arg Glu Pro Leu Ile Cys Pro Leu Cys Arg SerLys Trp Arg Ser His 290 295 300 gat ttc tac agc cac gag ttg tca agt cctgtg gat tcc cct tct tcc 959 Asp Phe Tyr Ser His Glu Leu Ser Ser Pro ValAsp Ser Pro Ser Ser 305 310 315 ctc aga gct gca cag cag caa acc gta cagcag cag cct ttg gct gga 1007 Leu Arg Ala Ala Gln Gln Gln Thr Val Gln GlnGln Pro Leu Ala Gly 320 325 330 335 tca cga agg aat caa gag agc aat tttaac ctt act cat tat gga act 1055 Ser Arg Arg Asn Gln Glu Ser Asn Phe AsnLeu Thr His Tyr Gly Thr 340 345 350 cag caa atc cct cct gct tac aaa gattta gct gag cca tgg att cag 1103 Gln Gln Ile Pro Pro Ala Tyr Lys Asp LeuAla Glu Pro Trp Ile Gln 355 360 365 gtg ttt gga atg gaa ctc gtt ggc tgctta ttc tct aga aac tgg aac 1151 Val Phe Gly Met Glu Leu Val Gly Cys LeuPhe Ser Arg Asn Trp Asn 370 375 380 gta agg gaa atg gcc ctt agg cgt ctttcc cac gac gtt agt ggg gcc 1199 Val Arg Glu Met Ala Leu Arg Arg Leu SerHis Asp Val Ser Gly Ala 385 390 395 ctg ttg ttg gca aac ggg gag agc actgga aac tct gga ggc ggc agt 1247 Leu Leu Leu Ala Asn Gly Glu Ser Thr GlyAsn Ser Gly Gly Gly Ser 400 405 410 415 ggg ggc agc tta agc gcg gga gcggcc agc ggg tcc tcc cag ccc agc 1295 Gly Gly Ser Leu Ser Ala Gly Ala AlaSer Gly Ser Ser Gln Pro Ser 420 425 430 atc tca ggg gat gtg gtg gag gcgtgc tgc agt gtc ctg tct ata gtc 1343 Ile Ser Gly Asp Val Val Glu Ala CysCys Ser Val Leu Ser Ile Val 435 440 445 tgc gct gac cct gtc tac aaa gtgtac gtt gct gct tta aaa aca ttg 1391 Cys Ala Asp Pro Val Tyr Lys Val TyrVal Ala Ala Leu Lys Thr Leu 450 455 460 aga gcc atg ctg gta tac act ccttgc cac agt ctg gca gaa aga atc 1439 Arg Ala Met Leu Val Tyr Thr Pro CysHis Ser Leu Ala Glu Arg Ile 465 470 475 aaa ctt cag aga ctc ctc cgg ccagtt gta gac act atc ctt gtc aag 1487 Lys Leu Gln Arg Leu Leu Arg Pro ValVal Asp Thr Ile Leu Val Lys 480 485 490 495 tgt gca gat gcc aac agc cgcacg agt cag ctg tcc ata tct aca gtg 1535 Cys Ala Asp Ala Asn Ser Arg ThrSer Gln Leu Ser Ile Ser Thr Val 500 505 510 ctg gaa ctc tgc aat ggc caagca gga aag ctg gcg gtt ggg aga gaa 1583 Leu Glu Leu Cys Asn Gly Gln AlaGly Lys Leu Ala Val Gly Arg Glu 515 520 525 ata ctt aaa gct ggg tcc atcggg gtt ggt ggt gtc gat tac gtc tta 1631 Ile Leu Lys Ala Gly Ser Ile GlyVal Gly Gly Val Asp Tyr Val Leu 530 535 540 agt tgt atc ctt gga aac caagct gaa tca aac aac tgg caa gaa ctg 1679 Ser Cys Ile Leu Gly Asn Gln AlaGlu Ser Asn Asn Trp Gln Glu Leu 545 550 555 ctg ggt cgc ctc tgt ctt atagac agg ttg ctg ttg gaa ttt cct gct 1727 Leu Gly Arg Leu Cys Leu Ile AspArg Leu Leu Leu Glu Phe Pro Ala 560 565 570 575 gaa ttc tat cct cat attgtc agt act gat gtc tca caa gct gag cct 1775 Glu Phe Tyr Pro His Ile ValSer Thr Asp Val Ser Gln Ala Glu Pro 580 585 590 gtt gaa atc agg tac aagaag ctg ctc tcc ctc tta acc ttt gcc ttg 1823 Val Glu Ile Arg Tyr Lys LysLeu Leu Ser Leu Leu Thr Phe Ala Leu 595 600 605 caa tcc att gac aat tcccac tcg atg gtt ggc aag ctc tct cgg agg 1871 Gln Ser Ile Asp Asn Ser HisSer Met Val Gly Lys Leu Ser Arg Arg 610 615 620 ata tat ctg agc tct gccagg atg gtg acc gca gtg ccc gct gtg ttt 1919 Ile Tyr Leu Ser Ser Ala ArgMet Val Thr Ala Val Pro Ala Val Phe 625 630 635 tcc aag ctg gta acc atgctt aat gct tct ggc tcc acc cac ttc acc 1967 Ser Lys Leu Val Thr Met LeuAsn Ala Ser Gly Ser Thr His Phe Thr 640 645 650 655 agg atg cgc cgg cgtctg atg gct atc gcg gat gag gta gaa att gcc 2015 Arg Met Arg Arg Arg LeuMet Ala Ile Ala Asp Glu Val Glu Ile Ala 660 665 670 gag gtc atc cag ctgggt gtg gag gac act gtg gat ggg cat cag gac 2063 Glu Val Ile Gln Leu GlyVal Glu Asp Thr Val Asp Gly His Gln Asp 675 680 685 agc tta cag gcg ctggcc ccc gcc agc tgt cta gaa aac agc tcc ctt 2111 Ser Leu Gln Ala Leu AlaPro Ala Ser Cys Leu Glu Asn Ser Ser Leu 690 695 700 gag cac aca gtc cataga gag aaa act gga aaa gga cta agt gct acg 2159 Glu His Thr Val His ArgGlu Lys Thr Gly Lys Gly Leu Ser Ala Thr 705 710 715 aga ctg agt gcc agctcg gag gac att tct gac aga ctg gcc ggc gtc 2207 Arg Leu Ser Ala Ser SerGlu Asp Ile Ser Asp Arg Leu Ala Gly Val 720 725 730 735 tct gta gga cttccc agc tca aca aca aca gaa caa cca aag cca gcg 2255 Ser Val Gly Leu ProSer Ser Thr Thr Thr Glu Gln Pro Lys Pro Ala 740 745 750 gtt caa aca aaaggc aga ccc cac agt cag tgt ttg aac tcc tcc cct 2303 Val Gln Thr Lys GlyArg Pro His Ser Gln Cys Leu Asn Ser Ser Pro 755 760 765 ttg tct cat gctcaa tta atg ttc cca gca cca tca gcc cct tgt tcc 2351 Leu Ser His Ala GlnLeu Met Phe Pro Ala Pro Ser Ala Pro Cys Ser 770 775 780 tct gcc ccg tctgtc cca gat att tct aag cac aga ccc cag gca ttt 2399 Ser Ala Pro Ser ValPro Asp Ile Ser Lys His Arg Pro Gln Ala Phe 785 790 795 gtt ccc tgc aaaata cct tcc gca tct cct cag aca cag cgc aag ttc 2447 Val Pro Cys Lys IlePro Ser Ala Ser Pro Gln Thr Gln Arg Lys Phe 800 805 810 815 tct cta caattc cag agg aac tgc tct gaa cac cga gac tca gac cag 2495 Ser Leu Gln PheGln Arg Asn Cys Ser Glu His Arg Asp Ser Asp Gln 820 825 830 ctc tcc ccagtc ttc act cag tca aga ccc cca ccc tcc agt aac ata 2543 Leu Ser Pro ValPhe Thr Gln Ser Arg Pro Pro Pro Ser Ser Asn Ile 835 840 845 cac agg ccaaag cca tcc cga ccc gtt ccg ggc agt aca agc aaa cta 2591 His Arg Pro LysPro Ser Arg Pro Val Pro Gly Ser Thr Ser Lys Leu 850 855 860 ggg gac gccaca aaa agt agc atg aca ctt gat ctg ggc agt gct tcc 2639 Gly Asp Ala ThrLys Ser Ser Met Thr Leu Asp Leu Gly Ser Ala Ser 865 870 875 agg tgt gacgac agc ttt ggc ggc ggc ggc aac agt ggc aac gcc gtc 2687 Arg Cys Asp AspSer Phe Gly Gly Gly Gly Asn Ser Gly Asn Ala Val 880 885 890 895 ata cccagc gac gag aca gtg ttc acg ccg gtg gag gac aag tgc agg 2735 Ile Pro SerAsp Glu Thr Val Phe Thr Pro Val Glu Asp Lys Cys Arg 900 905 910 tta gatgtg aac acc gag ctc aac tcc agc atc gag gac ctt ctt gaa 2783 Leu Asp ValAsn Thr Glu Leu Asn Ser Ser Ile Glu Asp Leu Leu Glu 915 920 925 gca tccatg cct tca agt gac acg aca gtc act ttc aag tcc gaa gtc 2831 Ala Ser MetPro Ser Ser Asp Thr Thr Val Thr Phe Lys Ser Glu Val 930 935 940 gcc gtcctc tct ccg gaa aag gcc gaa aat gac gac acc tac aaa gac 2879 Ala Val LeuSer Pro Glu Lys Ala Glu Asn Asp Asp Thr Tyr Lys Asp 945 950 955 gac gtcaat cat aat caa aag tgc aaa gaa aag atg gaa gct gaa gag 2927 Asp Val AsnHis Asn Gln Lys Cys Lys Glu Lys Met Glu Ala Glu Glu 960 965 970 975 gaggag gct tta gcg atc gcc atg gcg atg tca gcg tct cag gat gcc 2975 Glu GluAla Leu Ala Ile Ala Met Ala Met Ser Ala Ser Gln Asp Ala 980 985 990 ctcccc atc gtc cct cag ctg cag gtg gaa aat gga gaa gat att atc 3023 Leu ProIle Val Pro Gln Leu Gln Val Glu Asn Gly Glu Asp Ile Ile 995 1000 1005atc att cag cag gac aca cca gaa act ctt cca gga cat acc aaa gcg 3071 IleIle Gln Gln Asp Thr Pro Glu Thr Leu Pro Gly His Thr Lys Ala 1010 10151020 aaa cag cct tac aga gaa gac gct gag tgg ctg aaa ggc cag cag ata3119 Lys Gln Pro Tyr Arg Glu Asp Ala Glu Trp Leu Lys Gly Gln Gln Ile1025 1030 1035 ggc ctc gga gca ttt tct tct tgt tat cag gct caa gat gtggga act 3167 Gly Leu Gly Ala Phe Ser Ser Cys Tyr Gln Ala Gln Asp Val GlyThr 1040 1045 1050 1055 gga act tta atg gct gtt aaa cag gtg act tat gtcaga aac aca tct 3215 Gly Thr Leu Met Ala Val Lys Gln Val Thr Tyr Val ArgAsn Thr Ser 1060 1065 1070 tct gag caa gaa gaa gta gta gaa gca cta agagaa gag ata aga atg 3263 Ser Glu Gln Glu Glu Val Val Glu Ala Leu Arg GluGlu Ile Arg Met 1075 1080 1085 atg agc cat ctg aat cat cca aac atc attagg atg ttg gga gcc acg 3311 Met Ser His Leu Asn His Pro Asn Ile Ile ArgMet Leu Gly Ala Thr 1090 1095 1100 tgt gag aag agc aat tac aat ctc ttcatt gaa tgg atg gca ggg gga 3359 Cys Glu Lys Ser Asn Tyr Asn Leu Phe IleGlu Trp Met Ala Gly Gly 1105 1110 1115 tcg gtg gct cat ttg ctg agt aaatat gga gcc ttc aaa gaa tca gta 3407 Ser Val Ala His Leu Leu Ser Lys TyrGly Ala Phe Lys Glu Ser Val 1120 1125 1130 1135 gtt att aac tac act gaacag tta ctc cgt ggc ctt tcg tat ctc cat 3455 Val Ile Asn Tyr Thr Glu GlnLeu Leu Arg Gly Leu Ser Tyr Leu His 1140 1145 1150 gag aac cag atc attcac aga gat gtc aaa ggt gcc aat ttg ctc att 3503 Glu Asn Gln Ile Ile HisArg Asp Val Lys Gly Ala Asn Leu Leu Ile 1155 1160 1165 gac agc acc ggtcag agg ctg aga att gca gac ttt gga gct gca gcc 3551 Asp Ser Thr Gly GlnArg Leu Arg Ile Ala Asp Phe Gly Ala Ala Ala 1170 1175 1180 agg ttg gcatca aaa gga act ggt gca gga gag ttt cag gga caa tta 3599 Arg Leu Ala SerLys Gly Thr Gly Ala Gly Glu Phe Gln Gly Gln Leu 1185 1190 1195 ctg gggaca att gca ttc atg gcg cct gag gtc cta aga ggt cag cag 3647 Leu Gly ThrIle Ala Phe Met Ala Pro Glu Val Leu Arg Gly Gln Gln 1200 1205 1210 1215tat ggt agg agc tgt gat gta tgg agt gtt ggc tgc gcc att ata gaa 3695 TyrGly Arg Ser Cys Asp Val Trp Ser Val Gly Cys Ala Ile Ile Glu 1220 12251230 atg gct tgt gca aaa cca cct tgg aat gca gaa aaa cac tcc aat cat3743 Met Ala Cys Ala Lys Pro Pro Trp Asn Ala Glu Lys His Ser Asn His1235 1240 1245 ctc gcc ttg ata ttt aag att gct agc gca act act gca ccgtcc atc 3791 Leu Ala Leu Ile Phe Lys Ile Ala Ser Ala Thr Thr Ala Pro SerIle 1250 1255 1260 ccg tca cac ctg tcc cct ggt tta cga gat gtg gct cttcgt tgt tta 3839 Pro Ser His Leu Ser Pro Gly Leu Arg Asp Val Ala Leu ArgCys Leu 1265 1270 1275 gaa ctt cag cct cag gac cgg cct ccg tca aga gagctg ctg aaa cat 3887 Glu Leu Gln Pro Gln Asp Arg Pro Pro Ser Arg Glu LeuLeu Lys His 1280 1285 1290 1295 ccg gtc ttc cgt acc acg tgg tag 3911 ProVal Phe Arg Thr Thr Trp 1300 2 1302 PRT Homo sapiens 2 Ala Trp Lys HisGlu Trp Leu Glu Arg Arg Asn Arg Arg Gly Pro Val 1 5 10 15 Val Val LysPro Ile Pro Val Lys Gly Asp Gly Ser Glu Met Asn His 20 25 30 Leu Ala AlaGlu Ser Pro Gly Glu Val Gln Ala Ser Ala Ala Ser Pro 35 40 45 Ala Ser LysGly Arg Arg Ser Pro Ser Pro Gly Asn Ser Pro Ser Gly 50 55 60 Arg Thr ValLys Ser Glu Ser Pro Gly Val Arg Arg Lys Arg Val Ser 65 70 75 80 Pro ValPro Phe Gln Ser Gly Arg Ile Thr Pro Pro Arg Arg Ala Pro 85 90 95 Ser ProAsp Gly Phe Ser Pro Tyr Ser Pro Glu Glu Thr Asn Arg Arg 100 105 110 ValAsn Lys Val Met Arg Ala Arg Leu Tyr Leu Leu Gln Gln Ile Gly 115 120 125Pro Asn Ser Phe Leu Ile Gly Gly Asp Ser Pro Asp Asn Lys Tyr Arg 130 135140 Val Phe Ile Gly Pro Gln Asn Cys Ser Cys Ala Arg Gly Thr Phe Cys 145150 155 160 Ile His Leu Leu Phe Val Met Leu Arg Val Phe Gln Leu Glu ProSer 165 170 175 Asp Pro Met Leu Trp Arg Lys Thr Leu Lys Asn Phe Glu ValGlu Ser 180 185 190 Leu Phe Gln Lys Tyr His Ser Arg Arg Ser Ser Arg IleLys Ala Pro 195 200 205 Ser Arg Asn Thr Ile Gln Lys Phe Val Ser Arg MetSer Asn Ser His 210 215 220 Thr Leu Ser Ser Ser Ser Thr Ser Thr Ser SerSer Val Asn Ser Ile 225 230 235 240 Lys Asp Glu Glu Glu Gln Met Cys ProIle Cys Leu Leu Gly Met Leu 245 250 255 Asp Glu Glu Ser Leu Thr Val CysGlu Asp Gly Cys Arg Asn Lys Leu 260 265 270 His His His Cys Met Ser IleTrp Ala Glu Glu Cys Arg Arg Asn Arg 275 280 285 Glu Pro Leu Ile Cys ProLeu Cys Arg Ser Lys Trp Arg Ser His Asp 290 295 300 Phe Tyr Ser His GluLeu Ser Ser Pro Val Asp Ser Pro Ser Ser Leu 305 310 315 320 Arg Ala AlaGln Gln Gln Thr Val Gln Gln Gln Pro Leu Ala Gly Ser 325 330 335 Arg ArgAsn Gln Glu Ser Asn Phe Asn Leu Thr His Tyr Gly Thr Gln 340 345 350 GlnIle Pro Pro Ala Tyr Lys Asp Leu Ala Glu Pro Trp Ile Gln Val 355 360 365Phe Gly Met Glu Leu Val Gly Cys Leu Phe Ser Arg Asn Trp Asn Val 370 375380 Arg Glu Met Ala Leu Arg Arg Leu Ser His Asp Val Ser Gly Ala Leu 385390 395 400 Leu Leu Ala Asn Gly Glu Ser Thr Gly Asn Ser Gly Gly Gly SerGly 405 410 415 Gly Ser Leu Ser Ala Gly Ala Ala Ser Gly Ser Ser Gln ProSer Ile 420 425 430 Ser Gly Asp Val Val Glu Ala Cys Cys Ser Val Leu SerIle Val Cys 435 440 445 Ala Asp Pro Val Tyr Lys Val Tyr Val Ala Ala LeuLys Thr Leu Arg 450 455 460 Ala Met Leu Val Tyr Thr Pro Cys His Ser LeuAla Glu Arg Ile Lys 465 470 475 480 Leu Gln Arg Leu Leu Arg Pro Val ValAsp Thr Ile Leu Val Lys Cys 485 490 495 Ala Asp Ala Asn Ser Arg Thr SerGln Leu Ser Ile Ser Thr Val Leu 500 505 510 Glu Leu Cys Asn Gly Gln AlaGly Lys Leu Ala Val Gly Arg Glu Ile 515 520 525 Leu Lys Ala Gly Ser IleGly Val Gly Gly Val Asp Tyr Val Leu Ser 530 535 540 Cys Ile Leu Gly AsnGln Ala Glu Ser Asn Asn Trp Gln Glu Leu Leu 545 550 555 560 Gly Arg LeuCys Leu Ile Asp Arg Leu Leu Leu Glu Phe Pro Ala Glu 565 570 575 Phe TyrPro His Ile Val Ser Thr Asp Val Ser Gln Ala Glu Pro Val 580 585 590 GluIle Arg Tyr Lys Lys Leu Leu Ser Leu Leu Thr Phe Ala Leu Gln 595 600 605Ser Ile Asp Asn Ser His Ser Met Val Gly Lys Leu Ser Arg Arg Ile 610 615620 Tyr Leu Ser Ser Ala Arg Met Val Thr Ala Val Pro Ala Val Phe Ser 625630 635 640 Lys Leu Val Thr Met Leu Asn Ala Ser Gly Ser Thr His Phe ThrArg 645 650 655 Met Arg Arg Arg Leu Met Ala Ile Ala Asp Glu Val Glu IleAla Glu 660 665 670 Val Ile Gln Leu Gly Val Glu Asp Thr Val Asp Gly HisGln Asp Ser 675 680 685 Leu Gln Ala Leu Ala Pro Ala Ser Cys Leu Glu AsnSer Ser Leu Glu 690 695 700 His Thr Val His Arg Glu Lys Thr Gly Lys GlyLeu Ser Ala Thr Arg 705 710 715 720 Leu Ser Ala Ser Ser Glu Asp Ile SerAsp Arg Leu Ala Gly Val Ser 725 730 735 Val Gly Leu Pro Ser Ser Thr ThrThr Glu Gln Pro Lys Pro Ala Val 740 745 750 Gln Thr Lys Gly Arg Pro HisSer Gln Cys Leu Asn Ser Ser Pro Leu 755 760 765 Ser His Ala Gln Leu MetPhe Pro Ala Pro Ser Ala Pro Cys Ser Ser 770 775 780 Ala Pro Ser Val ProAsp Ile Ser Lys His Arg Pro Gln Ala Phe Val 785 790 795 800 Pro Cys LysIle Pro Ser Ala Ser Pro Gln Thr Gln Arg Lys Phe Ser 805 810 815 Leu GlnPhe Gln Arg Asn Cys Ser Glu His Arg Asp Ser Asp Gln Leu 820 825 830 SerPro Val Phe Thr Gln Ser Arg Pro Pro Pro Ser Ser Asn Ile His 835 840 845Arg Pro Lys Pro Ser Arg Pro Val Pro Gly Ser Thr Ser Lys Leu Gly 850 855860 Asp Ala Thr Lys Ser Ser Met Thr Leu Asp Leu Gly Ser Ala Ser Arg 865870 875 880 Cys Asp Asp Ser Phe Gly Gly Gly Gly Asn Ser Gly Asn Ala ValIle 885 890 895 Pro Ser Asp Glu Thr Val Phe Thr Pro Val Glu Asp Lys CysArg Leu 900 905 910 Asp Val Asn Thr Glu Leu Asn Ser Ser Ile Glu Asp LeuLeu Glu Ala 915 920 925 Ser Met Pro Ser Ser Asp Thr Thr Val Thr Phe LysSer Glu Val Ala 930 935 940 Val Leu Ser Pro Glu Lys Ala Glu Asn Asp AspThr Tyr Lys Asp Asp 945 950 955 960 Val Asn His Asn Gln Lys Cys Lys GluLys Met Glu Ala Glu Glu Glu 965 970 975 Glu Ala Leu Ala Ile Ala Met AlaMet Ser Ala Ser Gln Asp Ala Leu 980 985 990 Pro Ile Val Pro Gln Leu GlnVal Glu Asn Gly Glu Asp Ile Ile Ile 995 1000 1005 Ile Gln Gln Asp ThrPro Glu Thr Leu Pro Gly His Thr Lys Ala Lys 1010 1015 1020 Gln Pro TyrArg Glu Asp Ala Glu Trp Leu Lys Gly Gln Gln Ile Gly 1025 1030 1035 1040Leu Gly Ala Phe Ser Ser Cys Tyr Gln Ala Gln Asp Val Gly Thr Gly 10451050 1055 Thr Leu Met Ala Val Lys Gln Val Thr Tyr Val Arg Asn Thr SerSer 1060 1065 1070 Glu Gln Glu Glu Val Val Glu Ala Leu Arg Glu Glu IleArg Met Met 1075 1080 1085 Ser His Leu Asn His Pro Asn Ile Ile Arg MetLeu Gly Ala Thr Cys 1090 1095 1100 Glu Lys Ser Asn Tyr Asn Leu Phe IleGlu Trp Met Ala Gly Gly Ser 1105 1110 1115 1120 Val Ala His Leu Leu SerLys Tyr Gly Ala Phe Lys Glu Ser Val Val 1125 1130 1135 Ile Asn Tyr ThrGlu Gln Leu Leu Arg Gly Leu Ser Tyr Leu His Glu 1140 1145 1150 Asn GlnIle Ile His Arg Asp Val Lys Gly Ala Asn Leu Leu Ile Asp 1155 1160 1165Ser Thr Gly Gln Arg Leu Arg Ile Ala Asp Phe Gly Ala Ala Ala Arg 11701175 1180 Leu Ala Ser Lys Gly Thr Gly Ala Gly Glu Phe Gln Gly Gln LeuLeu 1185 1190 1195 1200 Gly Thr Ile Ala Phe Met Ala Pro Glu Val Leu ArgGly Gln Gln Tyr 1205 1210 1215 Gly Arg Ser Cys Asp Val Trp Ser Val GlyCys Ala Ile Ile Glu Met 1220 1225 1230 Ala Cys Ala Lys Pro Pro Trp AsnAla Glu Lys His Ser Asn His Leu 1235 1240 1245 Ala Leu Ile Phe Lys IleAla Ser Ala Thr Thr Ala Pro Ser Ile Pro 1250 1255 1260 Ser His Leu SerPro Gly Leu Arg Asp Val Ala Leu Arg Cys Leu Glu 1265 1270 1275 1280 LeuGln Pro Gln Asp Arg Pro Pro Ser Arg Glu Leu Leu Lys His Pro 1285 12901295 Val Phe Arg Thr Thr Trp 1300 3 2013 DNA Homo sapiens CDS(124)..(1980) 3 ggccagctcg gtggcctcct ctcggccctc ggctccgcga tccccgcccagcggccgggc 60 aataaagaat gttgatggga gaaccatttt cctaattttc aaattattgagctggtcgcc 120 ata atg gat gat cag caa gct ttg aat tca atc atg caa gatttg gct 168 Met Asp Asp Gln Gln Ala Leu Asn Ser Ile Met Gln Asp Leu Ala1 5 10 15 gtc ctt cat aag gcc agt cgg cca gca tta tct tta caa gaa accagg 216 Val Leu His Lys Ala Ser Arg Pro Ala Leu Ser Leu Gln Glu Thr Arg20 25 30 aaa gca aaa cct tca tca cca aaa aaa cag aat gat gtt cga gtc aaa264 Lys Ala Lys Pro Ser Ser Pro Lys Lys Gln Asn Asp Val Arg Val Lys 3540 45 ttt gaa cat aga gga gaa aaa agg atc ctg cag gtt act aga cca gtt312 Phe Glu His Arg Gly Glu Lys Arg Ile Leu Gln Val Thr Arg Pro Val 5055 60 aaa cta gaa gac ctg aga tct aag tct aag atc gcc ttt ggg cag tct360 Lys Leu Glu Asp Leu Arg Ser Lys Ser Lys Ile Ala Phe Gly Gln Ser 6570 75 atg gat cta cac tat acc aac aat gag ttg gta att ccg tta act acc408 Met Asp Leu His Tyr Thr Asn Asn Glu Leu Val Ile Pro Leu Thr Thr 8085 90 95 caa gat gac ttg gac aaa gct gtg gaa ctg ctg gat cgc agt att cac456 Gln Asp Asp Leu Asp Lys Ala Val Glu Leu Leu Asp Arg Ser Ile His 100105 110 atg aag agt ctc aag ata tta ctt gta gta aat ggg agt aca cag gct504 Met Lys Ser Leu Lys Ile Leu Leu Val Val Asn Gly Ser Thr Gln Ala 115120 125 act aat tta gaa cca tca ccg tca cca gaa gat ttg aat aat aca cca552 Thr Asn Leu Glu Pro Ser Pro Ser Pro Glu Asp Leu Asn Asn Thr Pro 130135 140 ctt ggt gca gag agg aaa aag cgg cta tct gta gta ggt ccc cct aat600 Leu Gly Ala Glu Arg Lys Lys Arg Leu Ser Val Val Gly Pro Pro Asn 145150 155 agg gat aga agt tcc cct cct cca gga tac att cca gac gag cta cac648 Arg Asp Arg Ser Ser Pro Pro Pro Gly Tyr Ile Pro Asp Glu Leu His 160165 170 175 cag att gcc cgg aat ggg tca ttc act agc atc aac agt gaa ggagag 696 Gln Ile Ala Arg Asn Gly Ser Phe Thr Ser Ile Asn Ser Glu Gly Glu180 185 190 ttc att cca gag agc atg gac caa atg ctg gat cca ttg tct ttaagc 744 Phe Ile Pro Glu Ser Met Asp Gln Met Leu Asp Pro Leu Ser Leu Ser195 200 205 agc cct gaa aat tct ggc tca gga agc tgt ccg tca ctt gat agtcct 792 Ser Pro Glu Asn Ser Gly Ser Gly Ser Cys Pro Ser Leu Asp Ser Pro210 215 220 ttg gat gga gaa agc tac cca aaa tca cgg atg cct agg gca cagagc 840 Leu Asp Gly Glu Ser Tyr Pro Lys Ser Arg Met Pro Arg Ala Gln Ser225 230 235 tac cca gat aat cat cag gag ttt aca gac tat gat aac ccc attttt 888 Tyr Pro Asp Asn His Gln Glu Phe Thr Asp Tyr Asp Asn Pro Ile Phe240 245 250 255 gag aaa ttt gga aaa gga gga aca tat cca aga agg tac cacgtt tcc 936 Glu Lys Phe Gly Lys Gly Gly Thr Tyr Pro Arg Arg Tyr His ValSer 260 265 270 tat cat cac cag gag tat aat gac ggt cgg aag act ttt ccaaga gct 984 Tyr His His Gln Glu Tyr Asn Asp Gly Arg Lys Thr Phe Pro ArgAla 275 280 285 aga agg acc cag ggc acc agt ttc cgg tct cct gtg agc ttcagt cct 1032 Arg Arg Thr Gln Gly Thr Ser Phe Arg Ser Pro Val Ser Phe SerPro 290 295 300 act gat cac tcc tta agc aat agt agt gga agc agt gtc tttacc cca 1080 Thr Asp His Ser Leu Ser Asn Ser Ser Gly Ser Ser Val Phe ThrPro 305 310 315 gag tat gac gac agt cga atg aga aga cgg ggg agt gac atagac aac 1128 Glu Tyr Asp Asp Ser Arg Met Arg Arg Arg Gly Ser Asp Ile AspAsn 320 325 330 335 cct act ttg act gtc aca gac atc agc cca cca tgc cgttca cct cga 1176 Pro Thr Leu Thr Val Thr Asp Ile Ser Pro Pro Cys Arg SerPro Arg 340 345 350 gct ccg acc aac tgg aga ctg ggc aag ctg ctt ggc caagga gat ttt 1224 Ala Pro Thr Asn Trp Arg Leu Gly Lys Leu Leu Gly Gln GlyAsp Phe 355 360 365 ggt agg gtc tac ctc tgc tat gat gtt gat acc gga agagag ctg gct 1272 Gly Arg Val Tyr Leu Cys Tyr Asp Val Asp Thr Gly Arg GluLeu Ala 370 375 380 gtt aag caa gtt cag ttt aac cct gag agc cca gag accagc aag gaa 1320 Val Lys Gln Val Gln Phe Asn Pro Glu Ser Pro Glu Thr SerLys Glu 385 390 395 gta aat gca ctt gag tgt gaa att cag ttg ttg aaa aacttg ttg cat 1368 Val Asn Ala Leu Glu Cys Glu Ile Gln Leu Leu Lys Asn LeuLeu His 400 405 410 415 gag cga att gtt cag tat tat ggc tgt ttg agg gatcct cag gag aaa 1416 Glu Arg Ile Val Gln Tyr Tyr Gly Cys Leu Arg Asp ProGln Glu Lys 420 425 430 aca ctt tcc atc ttt atg gag tat atg cca ggg ggttca att aag gac 1464 Thr Leu Ser Ile Phe Met Glu Tyr Met Pro Gly Gly SerIle Lys Asp 435 440 445 caa cta aaa gcc tac gga gct ctt act gag aac gtgacg agg aag tac 1512 Gln Leu Lys Ala Tyr Gly Ala Leu Thr Glu Asn Val ThrArg Lys Tyr 450 455 460 acc cgt cag att ctg gag ggg gtc cat tat ttg catagt aat atg att 1560 Thr Arg Gln Ile Leu Glu Gly Val His Tyr Leu His SerAsn Met Ile 465 470 475 gtc cat aga gat atc aaa gga gca aat atc tta agggat tcc aca ggc 1608 Val His Arg Asp Ile Lys Gly Ala Asn Ile Leu Arg AspSer Thr Gly 480 485 490 495 aat atc aag tta gga gac ttt ggg gct agt aaacgg ctt cag acc atc 1656 Asn Ile Lys Leu Gly Asp Phe Gly Ala Ser Lys ArgLeu Gln Thr Ile 500 505 510 tgt ctc tca ggc aca gga atg aag tct gtc acaggc acg cca tac tgg 1704 Cys Leu Ser Gly Thr Gly Met Lys Ser Val Thr GlyThr Pro Tyr Trp 515 520 525 atg agt cct gag gtc atc agt gga gaa ggc tatgga aga aaa gca gac 1752 Met Ser Pro Glu Val Ile Ser Gly Glu Gly Tyr GlyArg Lys Ala Asp 530 535 540 atc tgg agt gta gca tgt aga gtg gta gaa atgcta act gaa aag cca 1800 Ile Trp Ser Val Ala Cys Arg Val Val Glu Met LeuThr Glu Lys Pro 545 550 555 cct tgg gct gaa ttt gaa gca atg gct gcc atcttt aag atc gcc act 1848 Pro Trp Ala Glu Phe Glu Ala Met Ala Ala Ile PheLys Ile Ala Thr 560 565 570 575 cag cca acg aac cca aag ctg cca cct catgtc tca gac tat act cgg 1896 Gln Pro Thr Asn Pro Lys Leu Pro Pro His ValSer Asp Tyr Thr Arg 580 585 590 gac ttc ctc aaa cgg att ttt gta gag gccaaa ctt cga cct tca gcg 1944 Asp Phe Leu Lys Arg Ile Phe Val Glu Ala LysLeu Arg Pro Ser Ala 595 600 605 gag gag ctc ttg cgg cac atg ttt gtg cattat cac tagcatcggc 1990 Glu Glu Leu Leu Arg His Met Phe Val His Tyr His610 615 ggcttcggtc ctccaccatc tcc 2013 4 619 PRT Homo sapiens 4 Met AspAsp Gln Gln Ala Leu Asn Ser Ile Met Gln Asp Leu Ala Val 1 5 10 15 LeuHis Lys Ala Ser Arg Pro Ala Leu Ser Leu Gln Glu Thr Arg Lys 20 25 30 AlaLys Pro Ser Ser Pro Lys Lys Gln Asn Asp Val Arg Val Lys Phe 35 40 45 GluHis Arg Gly Glu Lys Arg Ile Leu Gln Val Thr Arg Pro Val Lys 50 55 60 LeuGlu Asp Leu Arg Ser Lys Ser Lys Ile Ala Phe Gly Gln Ser Met 65 70 75 80Asp Leu His Tyr Thr Asn Asn Glu Leu Val Ile Pro Leu Thr Thr Gln 85 90 95Asp Asp Leu Asp Lys Ala Val Glu Leu Leu Asp Arg Ser Ile His Met 100 105110 Lys Ser Leu Lys Ile Leu Leu Val Val Asn Gly Ser Thr Gln Ala Thr 115120 125 Asn Leu Glu Pro Ser Pro Ser Pro Glu Asp Leu Asn Asn Thr Pro Leu130 135 140 Gly Ala Glu Arg Lys Lys Arg Leu Ser Val Val Gly Pro Pro AsnArg 145 150 155 160 Asp Arg Ser Ser Pro Pro Pro Gly Tyr Ile Pro Asp GluLeu His Gln 165 170 175 Ile Ala Arg Asn Gly Ser Phe Thr Ser Ile Asn SerGlu Gly Glu Phe 180 185 190 Ile Pro Glu Ser Met Asp Gln Met Leu Asp ProLeu Ser Leu Ser Ser 195 200 205 Pro Glu Asn Ser Gly Ser Gly Ser Cys ProSer Leu Asp Ser Pro Leu 210 215 220 Asp Gly Glu Ser Tyr Pro Lys Ser ArgMet Pro Arg Ala Gln Ser Tyr 225 230 235 240 Pro Asp Asn His Gln Glu PheThr Asp Tyr Asp Asn Pro Ile Phe Glu 245 250 255 Lys Phe Gly Lys Gly GlyThr Tyr Pro Arg Arg Tyr His Val Ser Tyr 260 265 270 His His Gln Glu TyrAsn Asp Gly Arg Lys Thr Phe Pro Arg Ala Arg 275 280 285 Arg Thr Gln GlyThr Ser Phe Arg Ser Pro Val Ser Phe Ser Pro Thr 290 295 300 Asp His SerLeu Ser Asn Ser Ser Gly Ser Ser Val Phe Thr Pro Glu 305 310 315 320 TyrAsp Asp Ser Arg Met Arg Arg Arg Gly Ser Asp Ile Asp Asn Pro 325 330 335Thr Leu Thr Val Thr Asp Ile Ser Pro Pro Cys Arg Ser Pro Arg Ala 340 345350 Pro Thr Asn Trp Arg Leu Gly Lys Leu Leu Gly Gln Gly Asp Phe Gly 355360 365 Arg Val Tyr Leu Cys Tyr Asp Val Asp Thr Gly Arg Glu Leu Ala Val370 375 380 Lys Gln Val Gln Phe Asn Pro Glu Ser Pro Glu Thr Ser Lys GluVal 385 390 395 400 Asn Ala Leu Glu Cys Glu Ile Gln Leu Leu Lys Asn LeuLeu His Glu 405 410 415 Arg Ile Val Gln Tyr Tyr Gly Cys Leu Arg Asp ProGln Glu Lys Thr 420 425 430 Leu Ser Ile Phe Met Glu Tyr Met Pro Gly GlySer Ile Lys Asp Gln 435 440 445 Leu Lys Ala Tyr Gly Ala Leu Thr Glu AsnVal Thr Arg Lys Tyr Thr 450 455 460 Arg Gln Ile Leu Glu Gly Val His TyrLeu His Ser Asn Met Ile Val 465 470 475 480 His Arg Asp Ile Lys Gly AlaAsn Ile Leu Arg Asp Ser Thr Gly Asn 485 490 495 Ile Lys Leu Gly Asp PheGly Ala Ser Lys Arg Leu Gln Thr Ile Cys 500 505 510 Leu Ser Gly Thr GlyMet Lys Ser Val Thr Gly Thr Pro Tyr Trp Met 515 520 525 Ser Pro Glu ValIle Ser Gly Glu Gly Tyr Gly Arg Lys Ala Asp Ile 530 535 540 Trp Ser ValAla Cys Arg Val Val Glu Met Leu Thr Glu Lys Pro Pro 545 550 555 560 TrpAla Glu Phe Glu Ala Met Ala Ala Ile Phe Lys Ile Ala Thr Gln 565 570 575Pro Thr Asn Pro Lys Leu Pro Pro His Val Ser Asp Tyr Thr Arg Asp 580 585590 Phe Leu Lys Arg Ile Phe Val Glu Ala Lys Leu Arg Pro Ser Ala Glu 595600 605 Glu Leu Leu Arg His Met Phe Val His Tyr His 610 615 5 1935 DNAHomo sapiens CDS (25)..(1902) 5 accgccgcct ccgccatcgc cacc atg gat caacaa gag gca tta gac tcg 51 Met Asp Gln Gln Glu Ala Leu Asp Ser 1 5 atcatg aag gac ctg gtg gcc ctc cag atg agc cga cga acc cgg ttg 99 Ile MetLys Asp Leu Val Ala Leu Gln Met Ser Arg Arg Thr Arg Leu 10 15 20 25 tctgga tat gag acc atg agg aat aag gac aca ggt cac cca aac agg 147 Ser GlyTyr Glu Thr Met Arg Asn Lys Asp Thr Gly His Pro Asn Arg 30 35 40 cag agtgac gtc aga atc aag ttt gaa cac aat ggg gag aga cga att 195 Gln Ser AspVal Arg Ile Lys Phe Glu His Asn Gly Glu Arg Arg Ile 45 50 55 ata gca ttcagc cgg cct gtg aga tac gaa gat gtg gag cac aag gtg 243 Ile Ala Phe SerArg Pro Val Arg Tyr Glu Asp Val Glu His Lys Val 60 65 70 aca aca gtc tttggg cag tct ctt gat ttg cat tat atg aat aat gag 291 Thr Thr Val Phe GlyGln Ser Leu Asp Leu His Tyr Met Asn Asn Glu 75 80 85 ctc tcc atc ctg ttgaaa aac caa gat gat ctc gat aaa gcc att gac 339 Leu Ser Ile Leu Leu LysAsn Gln Asp Asp Leu Asp Lys Ala Ile Asp 90 95 100 105 att ttg gat agaagc tca agt atg aaa agc ctt agg ata cta ctg tta 387 Ile Leu Asp Arg SerSer Ser Met Lys Ser Leu Arg Ile Leu Leu Leu 110 115 120 tcc caa gac agatac cat act agt tcc tct ccc cac tct gga gtg tcc 435 Ser Gln Asp Arg TyrHis Thr Ser Ser Ser Pro His Ser Gly Val Ser 125 130 135 agg cag gtt cggatc aag cct tcc cag tct gca ggg gat ata aat acc 483 Arg Gln Val Arg IleLys Pro Ser Gln Ser Ala Gly Asp Ile Asn Thr 140 145 150 atc tac caa gctcct gag ccc aga agc agg cac ctg tct gtc agc tcc 531 Ile Tyr Gln Ala ProGlu Pro Arg Ser Arg His Leu Ser Val Ser Ser 155 160 165 cag aac cct ggccga agc tca cct ccc ccg gga tat gtt cct gag cgg 579 Gln Asn Pro Gly ArgSer Ser Pro Pro Pro Gly Tyr Val Pro Glu Arg 170 175 180 185 caa cag cacatt gcc cgg caa gga tcc tac acc agc atc aac agt gag 627 Gln Gln His IleAla Arg Gln Gly Ser Tyr Thr Ser Ile Asn Ser Glu 190 195 200 ggg gag ttcatc cca gag acc agc gag cag tgc atg ctg gat ccc ctg 675 Gly Glu Phe IlePro Glu Thr Ser Glu Gln Cys Met Leu Asp Pro Leu 205 210 215 agc agt gcagaa aat tcc ttg tct gga agc tgc caa tcc ttg gac agg 723 Ser Ser Ala GluAsn Ser Leu Ser Gly Ser Cys Gln Ser Leu Asp Arg 220 225 230 tca gca gacagc cca tcc ttc cgg aaa tca cga atg tcc cgt gcc cag 771 Ser Ala Asp SerPro Ser Phe Arg Lys Ser Arg Met Ser Arg Ala Gln 235 240 245 agc ttc cctgac aac aga cag gaa tac tca gat cgg gaa act cag ctt 819 Ser Phe Pro AspAsn Arg Gln Glu Tyr Ser Asp Arg Glu Thr Gln Leu 250 255 260 265 tat gacaaa ggg gtc aaa ggt gga acc tac ccc cgg cgc tac cac gtg 867 Tyr Asp LysGly Val Lys Gly Gly Thr Tyr Pro Arg Arg Tyr His Val 270 275 280 tct gtgcac cac aag gac tac agt gat ggc aga aga aca ttt ccc cga 915 Ser Val HisHis Lys Asp Tyr Ser Asp Gly Arg Arg Thr Phe Pro Arg 285 290 295 ata cggcgt cat caa ggc aac ttg ttc acc ctg gtg ccc tcc agc cgc 963 Ile Arg ArgHis Gln Gly Asn Leu Phe Thr Leu Val Pro Ser Ser Arg 300 305 310 tcc ctgagc aca aat ggc gag aac atg ggt ctg gct gtg caa tac ctg 1011 Ser Leu SerThr Asn Gly Glu Asn Met Gly Leu Ala Val Gln Tyr Leu 315 320 325 gac ccccgt ggg cgc ctg cgg agt gcg gac agc gag aat gcc ctc tct 1059 Asp Pro ArgGly Arg Leu Arg Ser Ala Asp Ser Glu Asn Ala Leu Ser 330 335 340 345 gtgcag gag agg aat gtg cca acc aag tct ccc agt gcc ccc atc aac 1107 Val GlnGlu Arg Asn Val Pro Thr Lys Ser Pro Ser Ala Pro Ile Asn 350 355 360 tggcgc cgg gga aag ctc ctg ggc cag ggt gcc ttc ggc agg gtc tat 1155 Trp ArgArg Gly Lys Leu Leu Gly Gln Gly Ala Phe Gly Arg Val Tyr 365 370 375 ttgtgc tat gac gtg gac acg gga cgt gaa ctt gct tcc aag cag gtc 1203 Leu CysTyr Asp Val Asp Thr Gly Arg Glu Leu Ala Ser Lys Gln Val 380 385 390 caattt gat cca gac agt cct gag aca agc aag gag gtg agt gct ctg 1251 Gln PheAsp Pro Asp Ser Pro Glu Thr Ser Lys Glu Val Ser Ala Leu 395 400 405 gagtgc gag atc cag ttg cta aag aac ttg cag cat gag cgc act gtg 1299 Glu CysGlu Ile Gln Leu Leu Lys Asn Leu Gln His Glu Arg Thr Val 410 415 420 425cag tac tac ggc tgc ctg cgg gac cgt act cag aag atc ctc acc atc 1347 GlnTyr Tyr Gly Cys Leu Arg Asp Arg Thr Gln Lys Ile Leu Thr Ile 430 435 440ttt atg gag tat atg cca ggg ggc tct gta aaa gac cag ttg aag gcc 1395 PheMet Glu Tyr Met Pro Gly Gly Ser Val Lys Asp Gln Leu Lys Ala 445 450 455tac gga gct ctg aca gag agt gtg acc cgc aag tac acc cgg cag att 1443 TyrGly Ala Leu Thr Glu Ser Val Thr Arg Lys Tyr Thr Arg Gln Ile 460 465 470ctg gag ggc atg tca tac ctg cac agc aac atg att gtg cat cgg gac 1491 LeuGlu Gly Met Ser Tyr Leu His Ser Asn Met Ile Val His Arg Asp 475 480 485atc aag gga gcc aat atc ctc cga gac tca gct ggg aat gtg aag ctt 1539 IleLys Gly Ala Asn Ile Leu Arg Asp Ser Ala Gly Asn Val Lys Leu 490 495 500505 ggg gat ttt ggg gcc agc aaa cac cta cag acc atc tgc atg tca ggg 1587Gly Asp Phe Gly Ala Ser Lys His Leu Gln Thr Ile Cys Met Ser Gly 510 515520 aca ggc att cgc tct gtc act gac aca ccc tac tgg atg agt cct gaa 1635Thr Gly Ile Arg Ser Val Thr Asp Thr Pro Tyr Trp Met Ser Pro Glu 525 530535 gtc atc agt ggc gag ggc tat gga aga aag gca gac gtg tgg agc ctg 1683Val Ile Ser Gly Glu Gly Tyr Gly Arg Lys Ala Asp Val Trp Ser Leu 540 545550 ggc tgt act gtg gtg gat atg ctg aca gag aaa cca cct tgg gca gag 1731Gly Cys Thr Val Val Asp Met Leu Thr Glu Lys Pro Pro Trp Ala Glu 555 560565 tat gaa gct atg gct gcc att ttc aag att gcc acc cag cct acc aat 1779Tyr Glu Ala Met Ala Ala Ile Phe Lys Ile Ala Thr Gln Pro Thr Asn 570 575580 585 cct cag ctg ccc tct cac atc tca gaa cac ggc agg gac ttc ctg agg1827 Pro Gln Leu Pro Ser His Ile Ser Glu His Gly Arg Asp Phe Leu Arg 590595 600 cgc ata ttt gtg gaa gct cgt cag aga ccc tca gcc gag gaa ctg ctc1875 Arg Ile Phe Val Glu Ala Arg Gln Arg Pro Ser Ala Glu Glu Leu Leu 605610 615 aca cac cac ttt aca cag cta gtg tac tgagctctca aggctatcag 1922Thr His His Phe Thr Gln Leu Val Tyr 620 625 gctgccagct gcc 1935 6 626PRT Homo sapiens 6 Met Asp Gln Gln Glu Ala Leu Asp Ser Ile Met Lys AspLeu Val Ala 1 5 10 15 Leu Gln Met Ser Arg Arg Thr Arg Leu Ser Gly TyrGlu Thr Met Arg 20 25 30 Asn Lys Asp Thr Gly His Pro Asn Arg Gln Ser AspVal Arg Ile Lys 35 40 45 Phe Glu His Asn Gly Glu Arg Arg Ile Ile Ala PheSer Arg Pro Val 50 55 60 Arg Tyr Glu Asp Val Glu His Lys Val Thr Thr ValPhe Gly Gln Ser 65 70 75 80 Leu Asp Leu His Tyr Met Asn Asn Glu Leu SerIle Leu Leu Lys Asn 85 90 95 Gln Asp Asp Leu Asp Lys Ala Ile Asp Ile LeuAsp Arg Ser Ser Ser 100 105 110 Met Lys Ser Leu Arg Ile Leu Leu Leu SerGln Asp Arg Tyr His Thr 115 120 125 Ser Ser Ser Pro His Ser Gly Val SerArg Gln Val Arg Ile Lys Pro 130 135 140 Ser Gln Ser Ala Gly Asp Ile AsnThr Ile Tyr Gln Ala Pro Glu Pro 145 150 155 160 Arg Ser Arg His Leu SerVal Ser Ser Gln Asn Pro Gly Arg Ser Ser 165 170 175 Pro Pro Pro Gly TyrVal Pro Glu Arg Gln Gln His Ile Ala Arg Gln 180 185 190 Gly Ser Tyr ThrSer Ile Asn Ser Glu Gly Glu Phe Ile Pro Glu Thr 195 200 205 Ser Glu GlnCys Met Leu Asp Pro Leu Ser Ser Ala Glu Asn Ser Leu 210 215 220 Ser GlySer Cys Gln Ser Leu Asp Arg Ser Ala Asp Ser Pro Ser Phe 225 230 235 240Arg Lys Ser Arg Met Ser Arg Ala Gln Ser Phe Pro Asp Asn Arg Gln 245 250255 Glu Tyr Ser Asp Arg Glu Thr Gln Leu Tyr Asp Lys Gly Val Lys Gly 260265 270 Gly Thr Tyr Pro Arg Arg Tyr His Val Ser Val His His Lys Asp Tyr275 280 285 Ser Asp Gly Arg Arg Thr Phe Pro Arg Ile Arg Arg His Gln GlyAsn 290 295 300 Leu Phe Thr Leu Val Pro Ser Ser Arg Ser Leu Ser Thr AsnGly Glu 305 310 315 320 Asn Met Gly Leu Ala Val Gln Tyr Leu Asp Pro ArgGly Arg Leu Arg 325 330 335 Ser Ala Asp Ser Glu Asn Ala Leu Ser Val GlnGlu Arg Asn Val Pro 340 345 350 Thr Lys Ser Pro Ser Ala Pro Ile Asn TrpArg Arg Gly Lys Leu Leu 355 360 365 Gly Gln Gly Ala Phe Gly Arg Val TyrLeu Cys Tyr Asp Val Asp Thr 370 375 380 Gly Arg Glu Leu Ala Ser Lys GlnVal Gln Phe Asp Pro Asp Ser Pro 385 390 395 400 Glu Thr Ser Lys Glu ValSer Ala Leu Glu Cys Glu Ile Gln Leu Leu 405 410 415 Lys Asn Leu Gln HisGlu Arg Thr Val Gln Tyr Tyr Gly Cys Leu Arg 420 425 430 Asp Arg Thr GlnLys Ile Leu Thr Ile Phe Met Glu Tyr Met Pro Gly 435 440 445 Gly Ser ValLys Asp Gln Leu Lys Ala Tyr Gly Ala Leu Thr Glu Ser 450 455 460 Val ThrArg Lys Tyr Thr Arg Gln Ile Leu Glu Gly Met Ser Tyr Leu 465 470 475 480His Ser Asn Met Ile Val His Arg Asp Ile Lys Gly Ala Asn Ile Leu 485 490495 Arg Asp Ser Ala Gly Asn Val Lys Leu Gly Asp Phe Gly Ala Ser Lys 500505 510 His Leu Gln Thr Ile Cys Met Ser Gly Thr Gly Ile Arg Ser Val Thr515 520 525 Asp Thr Pro Tyr Trp Met Ser Pro Glu Val Ile Ser Gly Glu GlyTyr 530 535 540 Gly Arg Lys Ala Asp Val Trp Ser Leu Gly Cys Thr Val ValAsp Met 545 550 555 560 Leu Thr Glu Lys Pro Pro Trp Ala Glu Tyr Glu AlaMet Ala Ala Ile 565 570 575 Phe Lys Ile Ala Thr Gln Pro Thr Asn Pro GlnLeu Pro Ser His Ile 580 585 590 Ser Glu His Gly Arg Asp Phe Leu Arg ArgIle Phe Val Glu Ala Arg 595 600 605 Gln Arg Pro Ser Ala Glu Glu Leu LeuThr His His Phe Thr Gln Leu 610 615 620 Val Tyr 625 7 5253 DNA Musmusculus CDS (15)..(4493) 7 gcccgcgaga gaaa atg gcg gcg gcg gcg ggc gatcgc gcc tcg tcg tcg 50 Met Ala Ala Ala Ala Gly Asp Arg Ala Ser Ser Ser 15 10 gga ttc ccg ggc gcc gcg gcg gcg agt ccc gag gcg ggc ggc ggc ggc 98Gly Phe Pro Gly Ala Ala Ala Ala Ser Pro Glu Ala Gly Gly Gly Gly 15 20 25gga gga gga gga gct ctc cag gga agc ggc gcg ccc gca gcg ggc gcg 146 GlyGly Gly Gly Ala Leu Gln Gly Ser Gly Ala Pro Ala Ala Gly Ala 30 35 40 gcgggg ctg ctg cgg gag cct ggc agc gcg ggc cgc gag cgc gcg gac 194 Ala GlyLeu Leu Arg Glu Pro Gly Ser Ala Gly Arg Glu Arg Ala Asp 45 50 55 60 tggcgg cgg cgg cac gtg cgc aaa gtg cgg agt gtg gag ctg gac cag 242 Trp ArgArg Arg His Val Arg Lys Val Arg Ser Val Glu Leu Asp Gln 65 70 75 ctg ccggag cag ccg ctc ttc ctc gcc gcc gcc tcg ccg ccc tgc cca 290 Leu Pro GluGln Pro Leu Phe Leu Ala Ala Ala Ser Pro Pro Cys Pro 80 85 90 tct act tccccg tcg ccg gag ccc gcg gac gcg gct gca gga gcg agt 338 Ser Thr Ser ProSer Pro Glu Pro Ala Asp Ala Ala Ala Gly Ala Ser 95 100 105 cgc ttc cagccc gcg gcg gga ccg cca ccc ccg gga gcg gcg agt cgc 386 Arg Phe Gln ProAla Ala Gly Pro Pro Pro Pro Gly Ala Ala Ser Arg 110 115 120 tgc ggc tcccac tct gcc gag ctg gcg gcc gcg cgg gac agc ggc gcc 434 Cys Gly Ser HisSer Ala Glu Leu Ala Ala Ala Arg Asp Ser Gly Ala 125 130 135 140 cgg agcccc gcg ggg gcg gag ccg ccc tct gca gcg gcc ccc tcc ggt 482 Arg Ser ProAla Gly Ala Glu Pro Pro Ser Ala Ala Ala Pro Ser Gly 145 150 155 cga gagatg gag aat aaa gaa acc ctc aaa gga ctg cac aag atg gag 530 Arg Glu MetGlu Asn Lys Glu Thr Leu Lys Gly Leu His Lys Met Glu 160 165 170 gat cgcccg gag gag aga atg atc cgg gag aag ctc aag gcg acc tgt 578 Asp Arg ProGlu Glu Arg Met Ile Arg Glu Lys Leu Lys Ala Thr Cys 175 180 185 atg ccggcc tgg aag cac gag tgg ttg gag agg agg aac agg aga ggc 626 Met Pro AlaTrp Lys His Glu Trp Leu Glu Arg Arg Asn Arg Arg Gly 190 195 200 cct gtggtg gtg aag cca atc cct att aaa gga gat gga tct gaa gtg 674 Pro Val ValVal Lys Pro Ile Pro Ile Lys Gly Asp Gly Ser Glu Val 205 210 215 220 aataac ttg gca gct gag ccc cag gga gag ggc cag gca ggt tcc gct 722 Asn AsnLeu Ala Ala Glu Pro Gln Gly Glu Gly Gln Ala Gly Ser Ala 225 230 235 gcacca gcc ccc aag ggc cga cga agc cca tct cct ggc agc tct ccg 770 Ala ProAla Pro Lys Gly Arg Arg Ser Pro Ser Pro Gly Ser Ser Pro 240 245 250 tcaggg cgc tcg gtg aag ccg gaa tcc cca gga gta aga cgg aaa cga 818 Ser GlyArg Ser Val Lys Pro Glu Ser Pro Gly Val Arg Arg Lys Arg 255 260 265 gtgtcc ccg gtg cct ttc cag agt ggc aga atc aca cca ccc cga aga 866 Val SerPro Val Pro Phe Gln Ser Gly Arg Ile Thr Pro Pro Arg Arg 270 275 280 gcccca tca ccg gat ggc ttc tcc ccg tac agc cca gag gag acg agc 914 Ala ProSer Pro Asp Gly Phe Ser Pro Tyr Ser Pro Glu Glu Thr Ser 285 290 295 300cgc cgc gtg aac aaa gtg atg aga gcc agg ctg tac ctg ctg cag cag 962 ArgArg Val Asn Lys Val Met Arg Ala Arg Leu Tyr Leu Leu Gln Gln 305 310 315ata gga ccc aac tct ttc ctg att gga gga gac agt cca gac aat aaa 1010 IleGly Pro Asn Ser Phe Leu Ile Gly Gly Asp Ser Pro Asp Asn Lys 320 325 330tac cgg gtg ttt att ggg cca cag aac tgc agc tgt ggg cgt gga gca 1058 TyrArg Val Phe Ile Gly Pro Gln Asn Cys Ser Cys Gly Arg Gly Ala 335 340 345ttc tgt att cac ctc ttg ttt gtc atg ctc cgg gtg ttt cag cta gaa 1106 PheCys Ile His Leu Leu Phe Val Met Leu Arg Val Phe Gln Leu Glu 350 355 360ccc tct gac ccc atg tta tgg aga aaa act tta aaa aat ttc gag gtt 1154 ProSer Asp Pro Met Leu Trp Arg Lys Thr Leu Lys Asn Phe Glu Val 365 370 375380 gag agt ttg ttc cag aaa tac cac agt agg cgt agc tcg aga atc aaa 1202Glu Ser Leu Phe Gln Lys Tyr His Ser Arg Arg Ser Ser Arg Ile Lys 385 390395 gct cca tcc cgg aac acc atc cag aag ttt gtg tca cgc atg tca aat 1250Ala Pro Ser Arg Asn Thr Ile Gln Lys Phe Val Ser Arg Met Ser Asn 400 405410 tct cac aca ctg tca tcg tct agc aca tcc aca tct agt tca gaa aac 1298Ser His Thr Leu Ser Ser Ser Ser Thr Ser Thr Ser Ser Ser Glu Asn 415 420425 agc atc aag gat gaa gag gag cag atg tgt ccc atc tgc ttg ctg ggc 1346Ser Ile Lys Asp Glu Glu Glu Gln Met Cys Pro Ile Cys Leu Leu Gly 430 435440 atg ctg gat gag gag agc ctg act gtg tgt gaa gat ggc tgc agg aac 1394Met Leu Asp Glu Glu Ser Leu Thr Val Cys Glu Asp Gly Cys Arg Asn 445 450455 460 aag ctg cac cac cat tgc atg tcc atc tgg gcg gaa gag tgt aga aga1442 Lys Leu His His His Cys Met Ser Ile Trp Ala Glu Glu Cys Arg Arg 465470 475 aat aga gag cct tta ata tgt ccc ctt tgt aga tct aag tgg aga tcc1490 Asn Arg Glu Pro Leu Ile Cys Pro Leu Cys Arg Ser Lys Trp Arg Ser 480485 490 cat gac ttc tac agc cat gag tta tca agc ccc gtg gag tcc ccc gcc1538 His Asp Phe Tyr Ser His Glu Leu Ser Ser Pro Val Glu Ser Pro Ala 495500 505 tcc ctg cga gct gtc cag cag cca tcc tcc ccg cag cag ccc gtg gcc1586 Ser Leu Arg Ala Val Gln Gln Pro Ser Ser Pro Gln Gln Pro Val Ala 510515 520 gga tca cag cgg agg aat cag gag agc agt ttt aac ctt act cat ttt1634 Gly Ser Gln Arg Arg Asn Gln Glu Ser Ser Phe Asn Leu Thr His Phe 525530 535 540 gga acc cag cag att cct tcc gct tac aaa gat ttg gcc gag ccatgg 1682 Gly Thr Gln Gln Ile Pro Ser Ala Tyr Lys Asp Leu Ala Glu Pro Trp545 550 555 att cag gtg ttt gga atg gaa ctc gtt ggc tgc tta ttc tct agaaac 1730 Ile Gln Val Phe Gly Met Glu Leu Val Gly Cys Leu Phe Ser Arg Asn560 565 570 tgg aac gta agg gaa atg gcc ctt agg cgt ctt tcc cac gac gttagt 1778 Trp Asn Val Arg Glu Met Ala Leu Arg Arg Leu Ser His Asp Val Ser575 580 585 ggg gcc ctg ttg ttg gca aac ggg gag agc act gga aac tct ggaggc 1826 Gly Ala Leu Leu Leu Ala Asn Gly Glu Ser Thr Gly Asn Ser Gly Gly590 595 600 ggc agt ggg ggc agc tta agc gcg gga gcg gcc agc ggg tcc tcccag 1874 Gly Ser Gly Gly Ser Leu Ser Ala Gly Ala Ala Ser Gly Ser Ser Gln605 610 615 620 ccc agc atc tca ggg gat gtg gtg gag gcg tgc tgc agt gtcctg tct 1922 Pro Ser Ile Ser Gly Asp Val Val Glu Ala Cys Cys Ser Val LeuSer 625 630 635 ata gtc tgc gct gac cct gtc tac aaa gtg tac gtt gct gcttta aaa 1970 Ile Val Cys Ala Asp Pro Val Tyr Lys Val Tyr Val Ala Ala LeuLys 640 645 650 aca ttg aga gcc atg ctg gta tac act cct tgc cac agt ctggca gaa 2018 Thr Leu Arg Ala Met Leu Val Tyr Thr Pro Cys His Ser Leu AlaGlu 655 660 665 aga atc aaa ctt cag aga ctc ctc cgg cca gtt gta gac actatc ctt 2066 Arg Ile Lys Leu Gln Arg Leu Leu Arg Pro Val Val Asp Thr IleLeu 670 675 680 gtc aag tgt gca gat gcc aac agc cgc acg agt cag ctg tccata tct 2114 Val Lys Cys Ala Asp Ala Asn Ser Arg Thr Ser Gln Leu Ser IleSer 685 690 695 700 aca gtg ctg gaa ctc tgc aag ggc caa gca gga gag ctggcg gtt ggg 2162 Thr Val Leu Glu Leu Cys Lys Gly Gln Ala Gly Glu Leu AlaVal Gly 705 710 715 aga gaa ata ctt aaa gct ggg tcc atc ggg gtt ggt ggtgtc gat tac 2210 Arg Glu Ile Leu Lys Ala Gly Ser Ile Gly Val Gly Gly ValAsp Tyr 720 725 730 gtc tta agt tgt atc ctt gga aac caa gct gaa tca aacaac tgg caa 2258 Val Leu Ser Cys Ile Leu Gly Asn Gln Ala Glu Ser Asn AsnTrp Gln 735 740 745 gaa ctg ctg ggt cgc ctc tgt ctt ata gac agg ttg ctgttg gaa ttt 2306 Glu Leu Leu Gly Arg Leu Cys Leu Ile Asp Arg Leu Leu LeuGlu Phe 750 755 760 cct gct gaa ttc tat cct cat att gtc agt act gat gtctca caa gct 2354 Pro Ala Glu Phe Tyr Pro His Ile Val Ser Thr Asp Val SerGln Ala 765 770 775 780 gag cct gtt gaa atc agg tac aag aag ctg ctc tccctc tta acc ttt 2402 Glu Pro Val Glu Ile Arg Tyr Lys Lys Leu Leu Ser LeuLeu Thr Phe 785 790 795 gcc ttg caa tcc att gac aat tcc cac tcg atg gttggc aag ctc tct 2450 Ala Leu Gln Ser Ile Asp Asn Ser His Ser Met Val GlyLys Leu Ser 800 805 810 cgg agg ata tat ctg agc tct gcc agg atg gtg accgca gtg ccc gct 2498 Arg Arg Ile Tyr Leu Ser Ser Ala Arg Met Val Thr AlaVal Pro Ala 815 820 825 gtg ttt tcc aag ctg gta acc atg ctt aat gct tctggc tcc acc cac 2546 Val Phe Ser Lys Leu Val Thr Met Leu Asn Ala Ser GlySer Thr His 830 835 840 ttc acc agg atg cgc cgg cgt ctg atg gct atc gcggat gag gta gaa 2594 Phe Thr Arg Met Arg Arg Arg Leu Met Ala Ile Ala AspGlu Val Glu 845 850 855 860 att gcc gag gtc atc cag ctg ggt gtg gag gacact gtg gat ggg cat 2642 Ile Ala Glu Val Ile Gln Leu Gly Val Glu Asp ThrVal Asp Gly His 865 870 875 cag gac agc tta cag gcc gtg gcc ccc acc agctgt cta gaa aac agc 2690 Gln Asp Ser Leu Gln Ala Val Ala Pro Thr Ser CysLeu Glu Asn Ser 880 885 890 tcc ctt gag cac aca gtc cat aga gag aaa actgga aaa gga cta agt 2738 Ser Leu Glu His Thr Val His Arg Glu Lys Thr GlyLys Gly Leu Ser 895 900 905 gct acg aga ctg agt gcc agc tcg gag gac atttct gac aga ctg gcc 2786 Ala Thr Arg Leu Ser Ala Ser Ser Glu Asp Ile SerAsp Arg Leu Ala 910 915 920 ggc gtc tct gta gga ctt ccc agc tca aca acaaca gaa caa cca aag 2834 Gly Val Ser Val Gly Leu Pro Ser Ser Thr Thr ThrGlu Gln Pro Lys 925 930 935 940 cca gcg gtt caa aca aaa ggc aga ccc cacagt cag tgt ttg aac tcc 2882 Pro Ala Val Gln Thr Lys Gly Arg Pro His SerGln Cys Leu Asn Ser 945 950 955 tcc cct ttg tct cat gct caa tta atg ttccca gca cca tca gcc cct 2930 Ser Pro Leu Ser His Ala Gln Leu Met Phe ProAla Pro Ser Ala Pro 960 965 970 tgt tcc tct gcc ccg tct gtc cca gat atttct aag cac aga ccc cag 2978 Cys Ser Ser Ala Pro Ser Val Pro Asp Ile SerLys His Arg Pro Gln 975 980 985 gca ttt gtt ccc tgc aaa ata cct tcc gcatct cct cag aca cag cgc 3026 Ala Phe Val Pro Cys Lys Ile Pro Ser Ala SerPro Gln Thr Gln Arg 990 995 1000 aag ttc tct cta caa ttc cag agg aac tgctct gaa cac cga gac tca 3074 Lys Phe Ser Leu Gln Phe Gln Arg Asn Cys SerGlu His Arg Asp Ser 1005 1010 1015 1020 gac cag ctc tcc cca gtc ttc actcag tca aga ccc cca ccc tcc agt 3122 Asp Gln Leu Ser Pro Val Phe Thr GlnSer Arg Pro Pro Pro Ser Ser 1025 1030 1035 aac ata cac agg cca aag ccatcc cga ccc gtt ccg ggc agt aca agc 3170 Asn Ile His Arg Pro Lys Pro SerArg Pro Val Pro Gly Ser Thr Ser 1040 1045 1050 aaa cta ggg gac gcc acaaaa agt agc atg aca ctt gat ctg ggc agt 3218 Lys Leu Gly Asp Ala Thr LysSer Ser Met Thr Leu Asp Leu Gly Ser 1055 1060 1065 gct tcc agg tgt gacgac agc ttt ggc ggc ggc ggc aac agt ggc aac 3266 Ala Ser Arg Cys Asp AspSer Phe Gly Gly Gly Gly Asn Ser Gly Asn 1070 1075 1080 gcc gtc ata cccagc gac gag aca gtg ttc acg ccg gtg gag gac aag 3314 Ala Val Ile Pro SerAsp Glu Thr Val Phe Thr Pro Val Glu Asp Lys 1085 1090 1095 1100 tgc aggtta gat gtg aac acc gag ctc aac tcc agc atc gag gac ctt 3362 Cys Arg LeuAsp Val Asn Thr Glu Leu Asn Ser Ser Ile Glu Asp Leu 1105 1110 1115 cttgaa gca tcc atg cct tca agt gac acg aca gtc act ttc aag tcc 3410 Leu GluAla Ser Met Pro Ser Ser Asp Thr Thr Val Thr Phe Lys Ser 1120 1125 1130gaa gtc gcc gtc ctc tct ccg gaa aag gcc gaa aat gac gac acc tac 3458 GluVal Ala Val Leu Ser Pro Glu Lys Ala Glu Asn Asp Asp Thr Tyr 1135 11401145 aaa gac gac gtc aat cat aat caa aag tgc aaa gaa aag atg gaa gct3506 Lys Asp Asp Val Asn His Asn Gln Lys Cys Lys Glu Lys Met Glu Ala1150 1155 1160 gaa gag gag gag gct tta gcg atc gcc atg gcg atg tca gcgtct cag 3554 Glu Glu Glu Glu Ala Leu Ala Ile Ala Met Ala Met Ser Ala SerGln 1165 1170 1175 1180 gat gcc ctc ccc atc gtc cct cag ctg cag gtg gaaaat gga gaa gat 3602 Asp Ala Leu Pro Ile Val Pro Gln Leu Gln Val Glu AsnGly Glu Asp 1185 1190 1195 att atc atc att cag cag gac aca cca gaa actctt cca gga cat acc 3650 Ile Ile Ile Ile Gln Gln Asp Thr Pro Glu Thr LeuPro Gly His Thr 1200 1205 1210 aaa gcg aaa cag cct tac aga gaa gac gctgag tgg ctg aaa ggc cag 3698 Lys Ala Lys Gln Pro Tyr Arg Glu Asp Ala GluTrp Leu Lys Gly Gln 1215 1220 1225 cag ata ggc ctc gga gca ttt tct tcctgt tac caa gca cag gat gtg 3746 Gln Ile Gly Leu Gly Ala Phe Ser Ser CysTyr Gln Ala Gln Asp Val 1230 1235 1240 ggg act ggg act tta atg gct gtgaaa cag gtg acg tac gtc aga aac 3794 Gly Thr Gly Thr Leu Met Ala Val LysGln Val Thr Tyr Val Arg Asn 1245 1250 1255 1260 aca tcc tcc gag cag gaggag gtg gtg gaa gcg ttg agg gaa gag atc 3842 Thr Ser Ser Glu Gln Glu GluVal Val Glu Ala Leu Arg Glu Glu Ile 1265 1270 1275 cgg atg atg ggt cacctc aac cat cca aac atc atc cgg atg ctg ggg 3890 Arg Met Met Gly His LeuAsn His Pro Asn Ile Ile Arg Met Leu Gly 1280 1285 1290 gcc acg tgc gagaag agc aac tac aac ctc ttc att gag tgg atg gcg 3938 Ala Thr Cys Glu LysSer Asn Tyr Asn Leu Phe Ile Glu Trp Met Ala 1295 1300 1305 gga gga tctgtg gct cac ctc ttg agt aaa tac gga gct ttc aag gag 3986 Gly Gly Ser ValAla His Leu Leu Ser Lys Tyr Gly Ala Phe Lys Glu 1310 1315 1320 tca gtcgtc att aac tac act gag cag tta ctg cgt ggc ctt tcc tat 4034 Ser Val ValIle Asn Tyr Thr Glu Gln Leu Leu Arg Gly Leu Ser Tyr 1325 1330 1335 1340ctc cac gag aac cag atc att cac aga gac gtc aaa ggt gcc aac ctg 4082 LeuHis Glu Asn Gln Ile Ile His Arg Asp Val Lys Gly Ala Asn Leu 1345 13501355 ctc att gac agc acc ggt cag agg ctg aga att gca gac ttt gga gct4130 Leu Ile Asp Ser Thr Gly Gln Arg Leu Arg Ile Ala Asp Phe Gly Ala1360 1365 1370 gct gcc agg ttg gca tca aaa gga acc ggt gca gga gag ttccag gga 4178 Ala Ala Arg Leu Ala Ser Lys Gly Thr Gly Ala Gly Glu Phe GlnGly 1375 1380 1385 cag tta ctg ggg aca att gca ttc atg gcg cct gag gtccta aga ggt 4226 Gln Leu Leu Gly Thr Ile Ala Phe Met Ala Pro Glu Val LeuArg Gly 1390 1395 1400 cag cag tat ggt agg agc tgt gat gta tgg agt gttggc tgc gcc att 4274 Gln Gln Tyr Gly Arg Ser Cys Asp Val Trp Ser Val GlyCys Ala Ile 1405 1410 1415 1420 ata gaa atg gct tgt gca aaa cca cct tggaat gca gaa aaa cac tcc 4322 Ile Glu Met Ala Cys Ala Lys Pro Pro Trp AsnAla Glu Lys His Ser 1425 1430 1435 aat cat ctc gcc ttg ata ttt aag attgct agc gca act act gca ccg 4370 Asn His Leu Ala Leu Ile Phe Lys Ile AlaSer Ala Thr Thr Ala Pro 1440 1445 1450 tcc atc ccg tca cac ctg tcc ccgggt ctg cgc gac gtg gcc gtg cgc 4418 Ser Ile Pro Ser His Leu Ser Pro GlyLeu Arg Asp Val Ala Val Arg 1455 1460 1465 tgc tta gaa ctt cag cct caggac cgg cct ccg tcc aga gag ctg ctg 4466 Cys Leu Glu Leu Gln Pro Gln AspArg Pro Pro Ser Arg Glu Leu Leu 1470 1475 1480 aaa cat ccg gtc ttc cgtacc acg tgg tagttaattg ttcagatcag 4513 Lys His Pro Val Phe Arg Thr ThrTrp 1485 1490 ctctaatgga gacaggatat gcaaccggga gagagaaaag agaacttgtgggcgaccatg 4573 ccgctaaccg cagccctcac gccactgaac agccagaaac ggggccagcggggaaccgta 4633 cctaagcatg tgattgacaa atcatgacct gtacctaagc tcgatatgcagacatctaca 4693 gctcgtgcag gaactgcaca ccgtgccttt cacaggactg gctctgggggaccaggaagg 4753 cgatggagtt tgcatgacta aagaacagaa gcataaattt atttttggagcactttttca 4813 gctaatcagt attaccatgt acatcaacat gcccgccaca tttcaaactcagactgtccc 4873 agatgtcaag atccactgtg tttgagtttg tttgcagttc cctcagcttgctggtaattg 4933 tggtgttttg ttttcgatgc aaatgtgatg taatattctt attttctttggatcaaagct 4993 ggactgaaaa ttgtactgtg taattatttt tgtgttttta atgttatttggtactcgaat 5053 tgtaaataac gtctactgct gtttattcca gtttctacta cctcaggtgtcctatagatt 5113 tttcttctac caaagttcac tctcagaatg aaattctacg tgctgtgtgactatgactcc 5173 taagacttcc agggcttaag ggctaactcc tattagcacc ttactatgtaagcaaatgct 5233 acaaaaaaaa aaaaaaaaaa 5253 8 1493 PRT Mus musculus 8 MetAla Ala Ala Ala Gly Asp Arg Ala Ser Ser Ser Gly Phe Pro Gly 1 5 10 15Ala Ala Ala Ala Ser Pro Glu Ala Gly Gly Gly Gly Gly Gly Gly Gly 20 25 30Ala Leu Gln Gly Ser Gly Ala Pro Ala Ala Gly Ala Ala Gly Leu Leu 35 40 45Arg Glu Pro Gly Ser Ala Gly Arg Glu Arg Ala Asp Trp Arg Arg Arg 50 55 60His Val Arg Lys Val Arg Ser Val Glu Leu Asp Gln Leu Pro Glu Gln 65 70 7580 Pro Leu Phe Leu Ala Ala Ala Ser Pro Pro Cys Pro Ser Thr Ser Pro 85 9095 Ser Pro Glu Pro Ala Asp Ala Ala Ala Gly Ala Ser Arg Phe Gln Pro 100105 110 Ala Ala Gly Pro Pro Pro Pro Gly Ala Ala Ser Arg Cys Gly Ser His115 120 125 Ser Ala Glu Leu Ala Ala Ala Arg Asp Ser Gly Ala Arg Ser ProAla 130 135 140 Gly Ala Glu Pro Pro Ser Ala Ala Ala Pro Ser Gly Arg GluMet Glu 145 150 155 160 Asn Lys Glu Thr Leu Lys Gly Leu His Lys Met GluAsp Arg Pro Glu 165 170 175 Glu Arg Met Ile Arg Glu Lys Leu Lys Ala ThrCys Met Pro Ala Trp 180 185 190 Lys His Glu Trp Leu Glu Arg Arg Asn ArgArg Gly Pro Val Val Val 195 200 205 Lys Pro Ile Pro Ile Lys Gly Asp GlySer Glu Val Asn Asn Leu Ala 210 215 220 Ala Glu Pro Gln Gly Glu Gly GlnAla Gly Ser Ala Ala Pro Ala Pro 225 230 235 240 Lys Gly Arg Arg Ser ProSer Pro Gly Ser Ser Pro Ser Gly Arg Ser 245 250 255 Val Lys Pro Glu SerPro Gly Val Arg Arg Lys Arg Val Ser Pro Val 260 265 270 Pro Phe Gln SerGly Arg Ile Thr Pro Pro Arg Arg Ala Pro Ser Pro 275 280 285 Asp Gly PheSer Pro Tyr Ser Pro Glu Glu Thr Ser Arg Arg Val Asn 290 295 300 Lys ValMet Arg Ala Arg Leu Tyr Leu Leu Gln Gln Ile Gly Pro Asn 305 310 315 320Ser Phe Leu Ile Gly Gly Asp Ser Pro Asp Asn Lys Tyr Arg Val Phe 325 330335 Ile Gly Pro Gln Asn Cys Ser Cys Gly Arg Gly Ala Phe Cys Ile His 340345 350 Leu Leu Phe Val Met Leu Arg Val Phe Gln Leu Glu Pro Ser Asp Pro355 360 365 Met Leu Trp Arg Lys Thr Leu Lys Asn Phe Glu Val Glu Ser LeuPhe 370 375 380 Gln Lys Tyr His Ser Arg Arg Ser Ser Arg Ile Lys Ala ProSer Arg 385 390 395 400 Asn Thr Ile Gln Lys Phe Val Ser Arg Met Ser AsnSer His Thr Leu 405 410 415 Ser Ser Ser Ser Thr Ser Thr Ser Ser Ser GluAsn Ser Ile Lys Asp 420 425 430 Glu Glu Glu Gln Met Cys Pro Ile Cys LeuLeu Gly Met Leu Asp Glu 435 440 445 Glu Ser Leu Thr Val Cys Glu Asp GlyCys Arg Asn Lys Leu His His 450 455 460 His Cys Met Ser Ile Trp Ala GluGlu Cys Arg Arg Asn Arg Glu Pro 465 470 475 480 Leu Ile Cys Pro Leu CysArg Ser Lys Trp Arg Ser His Asp Phe Tyr 485 490 495 Ser His Glu Leu SerSer Pro Val Glu Ser Pro Ala Ser Leu Arg Ala 500 505 510 Val Gln Gln ProSer Ser Pro Gln Gln Pro Val Ala Gly Ser Gln Arg 515 520 525 Arg Asn GlnGlu Ser Ser Phe Asn Leu Thr His Phe Gly Thr Gln Gln 530 535 540 Ile ProSer Ala Tyr Lys Asp Leu Ala Glu Pro Trp Ile Gln Val Phe 545 550 555 560Gly Met Glu Leu Val Gly Cys Leu Phe Ser Arg Asn Trp Asn Val Arg 565 570575 Glu Met Ala Leu Arg Arg Leu Ser His Asp Val Ser Gly Ala Leu Leu 580585 590 Leu Ala Asn Gly Glu Ser Thr Gly Asn Ser Gly Gly Gly Ser Gly Gly595 600 605 Ser Leu Ser Ala Gly Ala Ala Ser Gly Ser Ser Gln Pro Ser IleSer 610 615 620 Gly Asp Val Val Glu Ala Cys Cys Ser Val Leu Ser Ile ValCys Ala 625 630 635 640 Asp Pro Val Tyr Lys Val Tyr Val Ala Ala Leu LysThr Leu Arg Ala 645 650 655 Met Leu Val Tyr Thr Pro Cys His Ser Leu AlaGlu Arg Ile Lys Leu 660 665 670 Gln Arg Leu Leu Arg Pro Val Val Asp ThrIle Leu Val Lys Cys Ala 675 680 685 Asp Ala Asn Ser Arg Thr Ser Gln LeuSer Ile Ser Thr Val Leu Glu 690 695 700 Leu Cys Lys Gly Gln Ala Gly GluLeu Ala Val Gly Arg Glu Ile Leu 705 710 715 720 Lys Ala Gly Ser Ile GlyVal Gly Gly Val Asp Tyr Val Leu Ser Cys 725 730 735 Ile Leu Gly Asn GlnAla Glu Ser Asn Asn Trp Gln Glu Leu Leu Gly 740 745 750 Arg Leu Cys LeuIle Asp Arg Leu Leu Leu Glu Phe Pro Ala Glu Phe 755 760 765 Tyr Pro HisIle Val Ser Thr Asp Val Ser Gln Ala Glu Pro Val Glu 770 775 780 Ile ArgTyr Lys Lys Leu Leu Ser Leu Leu Thr Phe Ala Leu Gln Ser 785 790 795 800Ile Asp Asn Ser His Ser Met Val Gly Lys Leu Ser Arg Arg Ile Tyr 805 810815 Leu Ser Ser Ala Arg Met Val Thr Ala Val Pro Ala Val Phe Ser Lys 820825 830 Leu Val Thr Met Leu Asn Ala Ser Gly Ser Thr His Phe Thr Arg Met835 840 845 Arg Arg Arg Leu Met Ala Ile Ala Asp Glu Val Glu Ile Ala GluVal 850 855 860 Ile Gln Leu Gly Val Glu Asp Thr Val Asp Gly His Gln AspSer Leu 865 870 875 880 Gln Ala Val Ala Pro Thr Ser Cys Leu Glu Asn SerSer Leu Glu His 885 890 895 Thr Val His Arg Glu Lys Thr Gly Lys Gly LeuSer Ala Thr Arg Leu 900 905 910 Ser Ala Ser Ser Glu Asp Ile Ser Asp ArgLeu Ala Gly Val Ser Val 915 920 925 Gly Leu Pro Ser Ser Thr Thr Thr GluGln Pro Lys Pro Ala Val Gln 930 935 940 Thr Lys Gly Arg Pro His Ser GlnCys Leu Asn Ser Ser Pro Leu Ser 945 950 955 960 His Ala Gln Leu Met PhePro Ala Pro Ser Ala Pro Cys Ser Ser Ala 965 970 975 Pro Ser Val Pro AspIle Ser Lys His Arg Pro Gln Ala Phe Val Pro 980 985 990 Cys Lys Ile ProSer Ala Ser Pro Gln Thr Gln Arg Lys Phe Ser Leu 995 1000 1005 Gln PheGln Arg Asn Cys Ser Glu His Arg Asp Ser Asp Gln Leu Ser 1010 1015 1020Pro Val Phe Thr Gln Ser Arg Pro Pro Pro Ser Ser Asn Ile His Arg 10251030 1035 1040 Pro Lys Pro Ser Arg Pro Val Pro Gly Ser Thr Ser Lys LeuGly Asp 1045 1050 1055 Ala Thr Lys Ser Ser Met Thr Leu Asp Leu Gly SerAla Ser Arg Cys 1060 1065 1070 Asp Asp Ser Phe Gly Gly Gly Gly Asn SerGly Asn Ala Val Ile Pro 1075 1080 1085 Ser Asp Glu Thr Val Phe Thr ProVal Glu Asp Lys Cys Arg Leu Asp 1090 1095 1100 Val Asn Thr Glu Leu AsnSer Ser Ile Glu Asp Leu Leu Glu Ala Ser 1105 1110 1115 1120 Met Pro SerSer Asp Thr Thr Val Thr Phe Lys Ser Glu Val Ala Val 1125 1130 1135 LeuSer Pro Glu Lys Ala Glu Asn Asp Asp Thr Tyr Lys Asp Asp Val 1140 11451150 Asn His Asn Gln Lys Cys Lys Glu Lys Met Glu Ala Glu Glu Glu Glu1155 1160 1165 Ala Leu Ala Ile Ala Met Ala Met Ser Ala Ser Gln Asp AlaLeu Pro 1170 1175 1180 Ile Val Pro Gln Leu Gln Val Glu Asn Gly Glu AspIle Ile Ile Ile 1185 1190 1195 1200 Gln Gln Asp Thr Pro Glu Thr Leu ProGly His Thr Lys Ala Lys Gln 1205 1210 1215 Pro Tyr Arg Glu Asp Ala GluTrp Leu Lys Gly Gln Gln Ile Gly Leu 1220 1225 1230 Gly Ala Phe Ser SerCys Tyr Gln Ala Gln Asp Val Gly Thr Gly Thr 1235 1240 1245 Leu Met AlaVal Lys Gln Val Thr Tyr Val Arg Asn Thr Ser Ser Glu 1250 1255 1260 GlnGlu Glu Val Val Glu Ala Leu Arg Glu Glu Ile Arg Met Met Gly 1265 12701275 1280 His Leu Asn His Pro Asn Ile Ile Arg Met Leu Gly Ala Thr CysGlu 1285 1290 1295 Lys Ser Asn Tyr Asn Leu Phe Ile Glu Trp Met Ala GlyGly Ser Val 1300 1305 1310 Ala His Leu Leu Ser Lys Tyr Gly Ala Phe LysGlu Ser Val Val Ile 1315 1320 1325 Asn Tyr Thr Glu Gln Leu Leu Arg GlyLeu Ser Tyr Leu His Glu Asn 1330 1335 1340 Gln Ile Ile His Arg Asp ValLys Gly Ala Asn Leu Leu Ile Asp Ser 1345 1350 1355 1360 Thr Gly Gln ArgLeu Arg Ile Ala Asp Phe Gly Ala Ala Ala Arg Leu 1365 1370 1375 Ala SerLys Gly Thr Gly Ala Gly Glu Phe Gln Gly Gln Leu Leu Gly 1380 1385 1390Thr Ile Ala Phe Met Ala Pro Glu Val Leu Arg Gly Gln Gln Tyr Gly 13951400 1405 Arg Ser Cys Asp Val Trp Ser Val Gly Cys Ala Ile Ile Glu MetAla 1410 1415 1420 Cys Ala Lys Pro Pro Trp Asn Ala Glu Lys His Ser AsnHis Leu Ala 1425 1430 1435 1440 Leu Ile Phe Lys Ile Ala Ser Ala Thr ThrAla Pro Ser Ile Pro Ser 1445 1450 1455 His Leu Ser Pro Gly Leu Arg AspVal Ala Val Arg Cys Leu Glu Leu 1460 1465 1470 Gln Pro Gln Asp Arg ProPro Ser Arg Glu Leu Leu Lys His Pro Val 1475 1480 1485 Phe Arg Thr ThrTrp 1490 9 2465 DNA Mus musculus CDS (427)..(2283) 9 gaattcggcacgagggacga tccagcggca gagtcgccgc ttccgcttcg ctgcttctcc 60 ggtcaccggcgacgcgggcc cggggcttcc ttttcatcgg cccagcttat tccgcgggcc 120 ccggggctgcagctacccag aagcggcgaa gaggccctgg gctgcgcgcc cgctgtccca 180 tgtgaagcaggttgggcctg gtccccggcc cgtgcccggt tgtctgcggc ccttcaggcc 240 tcagggacccccgcgaggcg ctgctcctgg ggggcgcggt gacaggccgt gcgggggcgg 300 aggggccagctcggtggcct cctctcggcc ctcgcgtccg cgatcccgcc cagcggccgg 360 gcaataaagaatgttgatgg gagaaccatt ttcctaattt tcaaattatt gagctggtcg 420 cgcata atggat gat cag caa gct ttg aat tca atc atg caa gat ttg 468 Met Asp Asp GlnGln Ala Leu Asn Ser Ile Met Gln Asp Leu 1 5 10 gct gtc ctt cat aag ccagtc ggc cag cat tat ctt tac aag aaa cca 516 Ala Val Leu His Lys Pro ValGly Gln His Tyr Leu Tyr Lys Lys Pro 15 20 25 30 gga aag caa aac ctt catcac caa aaa aac aga atg atg ttc gag tca 564 Gly Lys Gln Asn Leu His HisGln Lys Asn Arg Met Met Phe Glu Ser 35 40 45 aat ttg aac ata gag gag gaaaaa agg atc ctg cag gtt act aga cca 612 Asn Leu Asn Ile Glu Glu Glu LysArg Ile Leu Gln Val Thr Arg Pro 50 55 60 gtt aaa cta gaa gac ctg aga tctaag tct aag atc gcc ttt ggg cag 660 Val Lys Leu Glu Asp Leu Arg Ser LysSer Lys Ile Ala Phe Gly Gln 65 70 75 tct atg gat cta cac tat acc aac aatgag ttg gta att ccg tta act 708 Ser Met Asp Leu His Tyr Thr Asn Asn GluLeu Val Ile Pro Leu Thr 80 85 90 acc caa gat gac ttg gac aaa gct gtg gaactg ctg gat cgc agt att 756 Thr Gln Asp Asp Leu Asp Lys Ala Val Glu LeuLeu Asp Arg Ser Ile 95 100 105 110 cac atg aag agt ctc aag ata tta cttgta gta aat ggg agt aca cag 804 His Met Lys Ser Leu Lys Ile Leu Leu ValVal Asn Gly Ser Thr Gln 115 120 125 gct act aat tta gaa cca tca ccg tcacca gaa gat ttg aat aat aca 852 Ala Thr Asn Leu Glu Pro Ser Pro Ser ProGlu Asp Leu Asn Asn Thr 130 135 140 cca ctt ggt gca gag agg aaa aag cggcta tct gta gta ggt ccc cct 900 Pro Leu Gly Ala Glu Arg Lys Lys Arg LeuSer Val Val Gly Pro Pro 145 150 155 aat agg gat aga agt tcc cct cct ccagga tac att cca gac ata cta 948 Asn Arg Asp Arg Ser Ser Pro Pro Pro GlyTyr Ile Pro Asp Ile Leu 160 165 170 cac cag att gcc cgg aat ggg tca ttcact agc atc aac agt gaa gga 996 His Gln Ile Ala Arg Asn Gly Ser Phe ThrSer Ile Asn Ser Glu Gly 175 180 185 190 gag ttc att cca gag agc atg gaccaa atg ctg gat cca ttg tct tta 1044 Glu Phe Ile Pro Glu Ser Met Asp GlnMet Leu Asp Pro Leu Ser Leu 195 200 205 agc agc cct gaa aat tct ggc tcagga agc tgt ccg tca ctt gat agt 1092 Ser Ser Pro Glu Asn Ser Gly Ser GlySer Cys Pro Ser Leu Asp Ser 210 215 220 cct ttg gat gga gaa agc tac ccaaaa tca cgg atg cct agg gca cag 1140 Pro Leu Asp Gly Glu Ser Tyr Pro LysSer Arg Met Pro Arg Ala Gln 225 230 235 agc tac cca gat aat cat cag gagttt aca gac tat gat aac ccc att 1188 Ser Tyr Pro Asp Asn His Gln Glu PheThr Asp Tyr Asp Asn Pro Ile 240 245 250 ttt gag aaa ttt gga aaa gga ggaaca tat cca aga agg tac cac gtt 1236 Phe Glu Lys Phe Gly Lys Gly Gly ThrTyr Pro Arg Arg Tyr His Val 255 260 265 270 tcc tat cat cac cag gag tataat gac ggt cgg aag act ttt cca aga 1284 Ser Tyr His His Gln Glu Tyr AsnAsp Gly Arg Lys Thr Phe Pro Arg 275 280 285 gct aga agg acc cag ggc accagt ttc cgg tct cct gtg agc ttc agt 1332 Ala Arg Arg Thr Gln Gly Thr SerPhe Arg Ser Pro Val Ser Phe Ser 290 295 300 cct act gat cac tcc tta agcact agt agt gga agc agt gtc ttt acc 1380 Pro Thr Asp His Ser Leu Ser ThrSer Ser Gly Ser Ser Val Phe Thr 305 310 315 cca gag tat gac gac agt cgaata aga aga cgg ggg agt gac ata gac 1428 Pro Glu Tyr Asp Asp Ser Arg IleArg Arg Arg Gly Ser Asp Ile Asp 320 325 330 aat cct act ttg act gtc acagac atc agc cca ccc agc cgt tca cct 1476 Asn Pro Thr Leu Thr Val Thr AspIle Ser Pro Pro Ser Arg Ser Pro 335 340 345 350 cga gct ccg acc aac tggaga ctg ggc aag ctg ctt ggc caa gga gct 1524 Arg Ala Pro Thr Asn Trp ArgLeu Gly Lys Leu Leu Gly Gln Gly Ala 355 360 365 ttt ggt agg gtc tac ctctgc tat gat gtt gat acc gga aga gag ctg 1572 Phe Gly Arg Val Tyr Leu CysTyr Asp Val Asp Thr Gly Arg Glu Leu 370 375 380 gct gtt aag caa gtt cagttt aac cct gag agc cca gag acc agc aag 1620 Ala Val Lys Gln Val Gln PheAsn Pro Glu Ser Pro Glu Thr Ser Lys 385 390 395 gaa gta aat gca ctt gagtgt gaa att cag ttg ttg aaa aac ttg ttg 1668 Glu Val Asn Ala Leu Glu CysGlu Ile Gln Leu Leu Lys Asn Leu Leu 400 405 410 cat gag cga att gtt cagtat tat ggc tgt ttg agg gat cct cag gag 1716 His Glu Arg Ile Val Gln TyrTyr Gly Cys Leu Arg Asp Pro Gln Glu 415 420 425 430 aaa aca ctt tcc atcttt atg gag ctc tcg cca ggg ggt tca att aag 1764 Lys Thr Leu Ser Ile PheMet Glu Leu Ser Pro Gly Gly Ser Ile Lys 435 440 445 gac caa cta aaa gcctac gga gct ctt act gag aac gtg acg agg aag 1812 Asp Gln Leu Lys Ala TyrGly Ala Leu Thr Glu Asn Val Thr Arg Lys 450 455 460 tac acc cgt cag attctg gag ggg gtc cat tat ttg cat agt aat atg 1860 Tyr Thr Arg Gln Ile LeuGlu Gly Val His Tyr Leu His Ser Asn Met 465 470 475 att gtc cat aga gatatc aaa gga gca aat atc tta agg gat tcc aca 1908 Ile Val His Arg Asp IleLys Gly Ala Asn Ile Leu Arg Asp Ser Thr 480 485 490 ggc aat atc aag ttagga gac ttt ggg gct agt aaa cgg ctt cag acc 1956 Gly Asn Ile Lys Leu GlyAsp Phe Gly Ala Ser Lys Arg Leu Gln Thr 495 500 505 510 atc tgt ctc tcaggc aca gga atg aag tct gtc aca ggc acg cca tac 2004 Ile Cys Leu Ser GlyThr Gly Met Lys Ser Val Thr Gly Thr Pro Tyr 515 520 525 tgg atg agt cctgag gtc atc agt gga gaa ggc tat gga aga aaa gca 2052 Trp Met Ser Pro GluVal Ile Ser Gly Glu Gly Tyr Gly Arg Lys Ala 530 535 540 gac atc tgg agtgta gca tgt act gtg gta gaa atg cta act gaa aag 2100 Asp Ile Trp Ser ValAla Cys Thr Val Val Glu Met Leu Thr Glu Lys 545 550 555 cca cct tgg gctgaa ttt gaa gca atg gct gcc atc ttt aag atc gcc 2148 Pro Pro Trp Ala GluPhe Glu Ala Met Ala Ala Ile Phe Lys Ile Ala 560 565 570 act cag cca acgaac cca aag ctg cca cct cat gtc tca gac tat act 2196 Thr Gln Pro Thr AsnPro Lys Leu Pro Pro His Val Ser Asp Tyr Thr 575 580 585 590 cgg gac ttcctc aaa cgg att ttt gta gag gcc aaa ctt cga cct tca 2244 Arg Asp Phe LeuLys Arg Ile Phe Val Glu Ala Lys Leu Arg Pro Ser 595 600 605 gcg gag gagctc ttg cgg cac atg ttt gtg cat tat cac tagcagcggc 2293 Ala Glu Glu LeuLeu Arg His Met Phe Val His Tyr His 610 615 ggcttcggtc ctccaccagctccatcctcg cggccacctt ctctcttact gcactttcct 2353 tttttataaa aaagagagatggggagaaaa agacaagagg gaaaatattt ctcttgattc 2413 ttggttaaat ttgtttaataataatagtaa actaaaaaaa aaaaaaaaaa aa 2465 10 619 PRT Mus musculus 10 MetAsp Asp Gln Gln Ala Leu Asn Ser Ile Met Gln Asp Leu Ala Val 1 5 10 15Leu His Lys Pro Val Gly Gln His Tyr Leu Tyr Lys Lys Pro Gly Lys 20 25 30Gln Asn Leu His His Gln Lys Asn Arg Met Met Phe Glu Ser Asn Leu 35 40 45Asn Ile Glu Glu Glu Lys Arg Ile Leu Gln Val Thr Arg Pro Val Lys 50 55 60Leu Glu Asp Leu Arg Ser Lys Ser Lys Ile Ala Phe Gly Gln Ser Met 65 70 7580 Asp Leu His Tyr Thr Asn Asn Glu Leu Val Ile Pro Leu Thr Thr Gln 85 9095 Asp Asp Leu Asp Lys Ala Val Glu Leu Leu Asp Arg Ser Ile His Met 100105 110 Lys Ser Leu Lys Ile Leu Leu Val Val Asn Gly Ser Thr Gln Ala Thr115 120 125 Asn Leu Glu Pro Ser Pro Ser Pro Glu Asp Leu Asn Asn Thr ProLeu 130 135 140 Gly Ala Glu Arg Lys Lys Arg Leu Ser Val Val Gly Pro ProAsn Arg 145 150 155 160 Asp Arg Ser Ser Pro Pro Pro Gly Tyr Ile Pro AspIle Leu His Gln 165 170 175 Ile Ala Arg Asn Gly Ser Phe Thr Ser Ile AsnSer Glu Gly Glu Phe 180 185 190 Ile Pro Glu Ser Met Asp Gln Met Leu AspPro Leu Ser Leu Ser Ser 195 200 205 Pro Glu Asn Ser Gly Ser Gly Ser CysPro Ser Leu Asp Ser Pro Leu 210 215 220 Asp Gly Glu Ser Tyr Pro Lys SerArg Met Pro Arg Ala Gln Ser Tyr 225 230 235 240 Pro Asp Asn His Gln GluPhe Thr Asp Tyr Asp Asn Pro Ile Phe Glu 245 250 255 Lys Phe Gly Lys GlyGly Thr Tyr Pro Arg Arg Tyr His Val Ser Tyr 260 265 270 His His Gln GluTyr Asn Asp Gly Arg Lys Thr Phe Pro Arg Ala Arg 275 280 285 Arg Thr GlnGly Thr Ser Phe Arg Ser Pro Val Ser Phe Ser Pro Thr 290 295 300 Asp HisSer Leu Ser Thr Ser Ser Gly Ser Ser Val Phe Thr Pro Glu 305 310 315 320Tyr Asp Asp Ser Arg Ile Arg Arg Arg Gly Ser Asp Ile Asp Asn Pro 325 330335 Thr Leu Thr Val Thr Asp Ile Ser Pro Pro Ser Arg Ser Pro Arg Ala 340345 350 Pro Thr Asn Trp Arg Leu Gly Lys Leu Leu Gly Gln Gly Ala Phe Gly355 360 365 Arg Val Tyr Leu Cys Tyr Asp Val Asp Thr Gly Arg Glu Leu AlaVal 370 375 380 Lys Gln Val Gln Phe Asn Pro Glu Ser Pro Glu Thr Ser LysGlu Val 385 390 395 400 Asn Ala Leu Glu Cys Glu Ile Gln Leu Leu Lys AsnLeu Leu His Glu 405 410 415 Arg Ile Val Gln Tyr Tyr Gly Cys Leu Arg AspPro Gln Glu Lys Thr 420 425 430 Leu Ser Ile Phe Met Glu Leu Ser Pro GlyGly Ser Ile Lys Asp Gln 435 440 445 Leu Lys Ala Tyr Gly Ala Leu Thr GluAsn Val Thr Arg Lys Tyr Thr 450 455 460 Arg Gln Ile Leu Glu Gly Val HisTyr Leu His Ser Asn Met Ile Val 465 470 475 480 His Arg Asp Ile Lys GlyAla Asn Ile Leu Arg Asp Ser Thr Gly Asn 485 490 495 Ile Lys Leu Gly AspPhe Gly Ala Ser Lys Arg Leu Gln Thr Ile Cys 500 505 510 Leu Ser Gly ThrGly Met Lys Ser Val Thr Gly Thr Pro Tyr Trp Met 515 520 525 Ser Pro GluVal Ile Ser Gly Glu Gly Tyr Gly Arg Lys Ala Asp Ile 530 535 540 Trp SerVal Ala Cys Thr Val Val Glu Met Leu Thr Glu Lys Pro Pro 545 550 555 560Trp Ala Glu Phe Glu Ala Met Ala Ala Ile Phe Lys Ile Ala Thr Gln 565 570575 Pro Thr Asn Pro Lys Leu Pro Pro His Val Ser Asp Tyr Thr Arg Asp 580585 590 Phe Leu Lys Arg Ile Phe Val Glu Ala Lys Leu Arg Pro Ser Ala Glu595 600 605 Glu Leu Leu Arg His Met Phe Val His Tyr His 610 615 11 3332DNA Mus musculus CDS (332)..(2209) 11 gaattcggca cgaggaacag tggccggtcggagcgtcttc tggacttcag gactcgcagg 60 cggcccggtc gagtggcgcc gccgaggccgggttgggccg agcctgggag cgccggggat 120 gtagcgggcc aacctgctca tgccacagcgcccggccgcg gccggagccg gagcctgggg 180 aggcggcggg ggccccgagc gcagcccacggcccccgcgc ggagccaggc ccgctgccgt 240 ccccgccgcc cgctcccccg gcatgcagccccggctgcgg aggtgacact tctgggctgt 300 agtcgccacc gccgcctccg ccatcgccac catg gat gaa caa gag gca tta 352 Met Asp Glu Gln Glu Ala Leu 1 5 gac tcgatc atg aag gac ctg gtg gcc ctc cag atg agc cga cga acc 400 Asp Ser IleMet Lys Asp Leu Val Ala Leu Gln Met Ser Arg Arg Thr 10 15 20 cgg ttg tctgga tat gag acc atg aag aat aag gac aca ggt cac cca 448 Arg Leu Ser GlyTyr Glu Thr Met Lys Asn Lys Asp Thr Gly His Pro 25 30 35 aac agg cag agtgac gtc aga atc aag ttt gaa cac aat ggg gag aga 496 Asn Arg Gln Ser AspVal Arg Ile Lys Phe Glu His Asn Gly Glu Arg 40 45 50 55 cga att ata gcattc agc cgg cct gtg aga tac gaa gat gtg gag cac 544 Arg Ile Ile Ala PheSer Arg Pro Val Arg Tyr Glu Asp Val Glu His 60 65 70 aag gtg aca aca gtcttt ggg cag cct ctt gat ttg cat tat atg aat 592 Lys Val Thr Thr Val PheGly Gln Pro Leu Asp Leu His Tyr Met Asn 75 80 85 aat gag ctc tcc atc ctgttg aaa aac caa gat gat ctc gat aaa gcc 640 Asn Glu Leu Ser Ile Leu LeuLys Asn Gln Asp Asp Leu Asp Lys Ala 90 95 100 att gac att ttg gat agaagc tca agt atg aaa agc ctt agg ata cta 688 Ile Asp Ile Leu Asp Arg SerSer Ser Met Lys Ser Leu Arg Ile Leu 105 110 115 ctg tta tcc caa gac agaaac cat act agt tcc tct ccc cac tct gga 736 Leu Leu Ser Gln Asp Arg AsnHis Thr Ser Ser Ser Pro His Ser Gly 120 125 130 135 gtg tcc agg cag gttcgg atc aag cct tcc cag tct gca ggg gat ata 784 Val Ser Arg Gln Val ArgIle Lys Pro Ser Gln Ser Ala Gly Asp Ile 140 145 150 aat acc atc tac caagct cct gag ccc aga agc agg cac ctg tct gtc 832 Asn Thr Ile Tyr Gln AlaPro Glu Pro Arg Ser Arg His Leu Ser Val 155 160 165 agc tcc cag aac cctggc cga agc tct cct ccc ccg gga tat gta cct 880 Ser Ser Gln Asn Pro GlyArg Ser Ser Pro Pro Pro Gly Tyr Val Pro 170 175 180 gag cga caa cag cacatt gcc cgg caa gga tcc tat acg agc atc aac 928 Glu Arg Gln Gln His IleAla Arg Gln Gly Ser Tyr Thr Ser Ile Asn 185 190 195 agc gaa ggt gaa ttcatc cca gag acc agc gaa cag tgt atg cta gat 976 Ser Glu Gly Glu Phe IlePro Glu Thr Ser Glu Gln Cys Met Leu Asp 200 205 210 215 ccc ctc agc agtgcc gaa aat tcc ttg tca gga agc tgc caa tcc ttg 1024 Pro Leu Ser Ser AlaGlu Asn Ser Leu Ser Gly Ser Cys Gln Ser Leu 220 225 230 gac agg tca gcagac agc cca tcc ttc agg aaa tca caa atg tcc cga 1072 Asp Arg Ser Ala AspSer Pro Ser Phe Arg Lys Ser Gln Met Ser Arg 235 240 245 gcc cgg agc ttccca gac aac aga aag gaa tgc tca gat cgg gag acc 1120 Ala Arg Ser Phe ProAsp Asn Arg Lys Glu Cys Ser Asp Arg Glu Thr 250 255 260 cag ctc tat gataaa ggt gtc aaa ggt gga acc tat ccc agg cgc tac 1168 Gln Leu Tyr Asp LysGly Val Lys Gly Gly Thr Tyr Pro Arg Arg Tyr 265 270 275 cat gtg tct gtgcat cac aaa gac tac aat gat ggc aga aga aca ttt 1216 His Val Ser Val HisHis Lys Asp Tyr Asn Asp Gly Arg Arg Thr Phe 280 285 290 295 ccc cga atacga cgg cat caa ggc aac cta ttc act ctg gtg ccc tca 1264 Pro Arg Ile ArgArg His Gln Gly Asn Leu Phe Thr Leu Val Pro Ser 300 305 310 agt cgc tccttg agc aca aat ggc gag aac atg ggt gta gct gtg caa 1312 Ser Arg Ser LeuSer Thr Asn Gly Glu Asn Met Gly Val Ala Val Gln 315 320 325 tac ctg gacccc cgt ggg cgc cta cgg agt gca gac agt gag aat gcc 1360 Tyr Leu Asp ProArg Gly Arg Leu Arg Ser Ala Asp Ser Glu Asn Ala 330 335 340 ctc act gtgcag gaa agg aat gtg cca acc aaa tct cct agt gct ccc 1408 Leu Thr Val GlnGlu Arg Asn Val Pro Thr Lys Ser Pro Ser Ala Pro 345 350 355 atc aat tggcgt cgg ggg aag ctc ctg ggt caa ggt gcc ttc ggc agg 1456 Ile Asn Trp ArgArg Gly Lys Leu Leu Gly Gln Gly Ala Phe Gly Arg 360 365 370 375 gtc tacttg tgc tat gat gtg gac aca gga cgt gaa ctt gct tct aag 1504 Val Tyr LeuCys Tyr Asp Val Asp Thr Gly Arg Glu Leu Ala Ser Lys 380 385 390 cag gtccag ttt gac cca gat agt cct gag aca agc aag gag gtg agt 1552 Gln Val GlnPhe Asp Pro Asp Ser Pro Glu Thr Ser Lys Glu Val Ser 395 400 405 gct ctggag tgt gag atc cag ttg ctg aag aac ctg cag cat gag cgc 1600 Ala Leu GluCys Glu Ile Gln Leu Leu Lys Asn Leu Gln His Glu Arg 410 415 420 att gtgcag tac tac ggc tgc ctg cgg gac cgt gct gag aag atc ctc 1648 Ile Val GlnTyr Tyr Gly Cys Leu Arg Asp Arg Ala Glu Lys Ile Leu 425 430 435 acc atcttt atg gag tat atg cca ggg ggc tct gta aaa gac cag ttg 1696 Thr Ile PheMet Glu Tyr Met Pro Gly Gly Ser Val Lys Asp Gln Leu 440 445 450 455 aaggcc tac gga gct ctg aca gag agt gtg acc cgc aag tac acc cgg 1744 Lys AlaTyr Gly Ala Leu Thr Glu Ser Val Thr Arg Lys Tyr Thr Arg 460 465 470 cagatt ctg gag ggc atg tca tac ctg cac agc aac atg att gtg cat 1792 Gln IleLeu Glu Gly Met Ser Tyr Leu His Ser Asn Met Ile Val His 475 480 485 cgggac atc aag gga gcc aat atc ctc cga gac tca gct ggg aat gtg 1840 Arg AspIle Lys Gly Ala Asn Ile Leu Arg Asp Ser Ala Gly Asn Val 490 495 500 aagctt ggg gat ttt ggg gcc agc aaa cgc cta cag acc atc tgc atg 1888 Lys LeuGly Asp Phe Gly Ala Ser Lys Arg Leu Gln Thr Ile Cys Met 505 510 515 tcaggg aca ggc att cgc tct gtc act ggc aca ccc tac tgg atg agt 1936 Ser GlyThr Gly Ile Arg Ser Val Thr Gly Thr Pro Tyr Trp Met Ser 520 525 530 535cct gaa gtc atc agt ggc gag ggc tat gga aga aag gca gac gtg tgg 1984 ProGlu Val Ile Ser Gly Glu Gly Tyr Gly Arg Lys Ala Asp Val Trp 540 545 550agc ctg ggc tgt act gtg gtg gaa atg ctg aca gag aaa cca cct tgg 2032 SerLeu Gly Cys Thr Val Val Glu Met Leu Thr Glu Lys Pro Pro Trp 555 560 565gca gag tat gaa gct atg gct gcc att ttc aag att gcc acc cag cct 2080 AlaGlu Tyr Glu Ala Met Ala Ala Ile Phe Lys Ile Ala Thr Gln Pro 570 575 580acc aat cct cag ctg ccc tct cac atc tca gaa cac ggc agg gac ttc 2128 ThrAsn Pro Gln Leu Pro Ser His Ile Ser Glu His Gly Arg Asp Phe 585 590 595ctg agg cgc ata ttt gtg gaa gct cgt cag aga ccc tca gct gag gag 2176 LeuArg Arg Ile Phe Val Glu Ala Arg Gln Arg Pro Ser Ala Glu Glu 600 605 610615 ctg ctc aca cac cac ttt gca cag cta gtg tac tgagctctca aggctatcag2229 Leu Leu Thr His His Phe Ala Gln Leu Val Tyr 620 625 gctgccagctgccacctgct gagcaggcaa ggggctgctg tcaggctcag tgaagttgct 2289 gcttcttccaggcaaggcta tgaccagtgg agcatcggtc cagccattgt ttgtctgtgc 2349 cccatctgccactgggactc aaagccagga tgggatagct ctggcatcaa gactgggagc 2409 tccagcctgtaagacccaag agctttagca ccttaagctc agtatggcgg gaagggctgg 2469 aaacagtatgcaagactgcc atgggtcctg cctaccctca gatgtgtcct aacactgcag 2529 acagcactgaagtcaagagg gactggggca caggaggtcc tcaagggtat gaatagtgtt 2589 acttcattcagagtgttact ttgtttctct cccaatgttt ggagaccacc agcctgtctc 2649 tgggctgcaagcctgaggta aagcccagca tcccccagcc aacagaaggt agaggtttgg 2709 gctaccccactatagcttcc aggtattcgg tgtcagtcct gtcttaccaa agatgaatga 2769 agcaaatgttacactgcctt attctgggaa ggaggagcta ctcggataag cagggcctga 2829 gagatggagctgcctccaga aactggggag acccagtctt gtcaatgcaa ttgtctctgt 2889 tttacaagttggagtcactc ttatgctgtt cccagtttta aaactggaga ctttgccctc 2949 tgagctctggagacccatgt gggcttaggc ttggactgga tggaagagct gatggcctct 3009 gccccctggcctgccctctg ttccctcact ggagcagaga aagtagacaa cacaagtcag 3069 ggcacctggttctgggcagc tcagcagagt gcagggggtt gtctcaggct gtctgcatct 3129 caaatctgtcaggcctgagc ccactccatg ggaaagtcct tgagctgcca caaccgtgtc 3189 caaagccaccagctgtgttc ctcagcccga cctgtccact tgtcatcaac ctcattccct 3249 tcttgttcctcccacaaagg aggatgccag taggggctag ggaaagagtt atcattaaag 3309 gaaaggaaaaaaaaaaaaaa aaa 3332 12 626 PRT Mus musculus 12 Met Asp Glu Gln Glu AlaLeu Asp Ser Ile Met Lys Asp Leu Val Ala 1 5 10 15 Leu Gln Met Ser ArgArg Thr Arg Leu Ser Gly Tyr Glu Thr Met Lys 20 25 30 Asn Lys Asp Thr GlyHis Pro Asn Arg Gln Ser Asp Val Arg Ile Lys 35 40 45 Phe Glu His Asn GlyGlu Arg Arg Ile Ile Ala Phe Ser Arg Pro Val 50 55 60 Arg Tyr Glu Asp ValGlu His Lys Val Thr Thr Val Phe Gly Gln Pro 65 70 75 80 Leu Asp Leu HisTyr Met Asn Asn Glu Leu Ser Ile Leu Leu Lys Asn 85 90 95 Gln Asp Asp LeuAsp Lys Ala Ile Asp Ile Leu Asp Arg Ser Ser Ser 100 105 110 Met Lys SerLeu Arg Ile Leu Leu Leu Ser Gln Asp Arg Asn His Thr 115 120 125 Ser SerSer Pro His Ser Gly Val Ser Arg Gln Val Arg Ile Lys Pro 130 135 140 SerGln Ser Ala Gly Asp Ile Asn Thr Ile Tyr Gln Ala Pro Glu Pro 145 150 155160 Arg Ser Arg His Leu Ser Val Ser Ser Gln Asn Pro Gly Arg Ser Ser 165170 175 Pro Pro Pro Gly Tyr Val Pro Glu Arg Gln Gln His Ile Ala Arg Gln180 185 190 Gly Ser Tyr Thr Ser Ile Asn Ser Glu Gly Glu Phe Ile Pro GluThr 195 200 205 Ser Glu Gln Cys Met Leu Asp Pro Leu Ser Ser Ala Glu AsnSer Leu 210 215 220 Ser Gly Ser Cys Gln Ser Leu Asp Arg Ser Ala Asp SerPro Ser Phe 225 230 235 240 Arg Lys Ser Gln Met Ser Arg Ala Arg Ser PhePro Asp Asn Arg Lys 245 250 255 Glu Cys Ser Asp Arg Glu Thr Gln Leu TyrAsp Lys Gly Val Lys Gly 260 265 270 Gly Thr Tyr Pro Arg Arg Tyr His ValSer Val His His Lys Asp Tyr 275 280 285 Asn Asp Gly Arg Arg Thr Phe ProArg Ile Arg Arg His Gln Gly Asn 290 295 300 Leu Phe Thr Leu Val Pro SerSer Arg Ser Leu Ser Thr Asn Gly Glu 305 310 315 320 Asn Met Gly Val AlaVal Gln Tyr Leu Asp Pro Arg Gly Arg Leu Arg 325 330 335 Ser Ala Asp SerGlu Asn Ala Leu Thr Val Gln Glu Arg Asn Val Pro 340 345 350 Thr Lys SerPro Ser Ala Pro Ile Asn Trp Arg Arg Gly Lys Leu Leu 355 360 365 Gly GlnGly Ala Phe Gly Arg Val Tyr Leu Cys Tyr Asp Val Asp Thr 370 375 380 GlyArg Glu Leu Ala Ser Lys Gln Val Gln Phe Asp Pro Asp Ser Pro 385 390 395400 Glu Thr Ser Lys Glu Val Ser Ala Leu Glu Cys Glu Ile Gln Leu Leu 405410 415 Lys Asn Leu Gln His Glu Arg Ile Val Gln Tyr Tyr Gly Cys Leu Arg420 425 430 Asp Arg Ala Glu Lys Ile Leu Thr Ile Phe Met Glu Tyr Met ProGly 435 440 445 Gly Ser Val Lys Asp Gln Leu Lys Ala Tyr Gly Ala Leu ThrGlu Ser 450 455 460 Val Thr Arg Lys Tyr Thr Arg Gln Ile Leu Glu Gly MetSer Tyr Leu 465 470 475 480 His Ser Asn Met Ile Val His Arg Asp Ile LysGly Ala Asn Ile Leu 485 490 495 Arg Asp Ser Ala Gly Asn Val Lys Leu GlyAsp Phe Gly Ala Ser Lys 500 505 510 Arg Leu Gln Thr Ile Cys Met Ser GlyThr Gly Ile Arg Ser Val Thr 515 520 525 Gly Thr Pro Tyr Trp Met Ser ProGlu Val Ile Ser Gly Glu Gly Tyr 530 535 540 Gly Arg Lys Ala Asp Val TrpSer Leu Gly Cys Thr Val Val Glu Met 545 550 555 560 Leu Thr Glu Lys ProPro Trp Ala Glu Tyr Glu Ala Met Ala Ala Ile 565 570 575 Phe Lys Ile AlaThr Gln Pro Thr Asn Pro Gln Leu Pro Ser His Ile 580 585 590 Ser Glu HisGly Arg Asp Phe Leu Arg Arg Ile Phe Val Glu Ala Arg 595 600 605 Gln ArgPro Ser Ala Glu Glu Leu Leu Thr His His Phe Ala Gln Leu 610 615 620 ValTyr 625 13 1597 PRT Mus musculus 13 Met Arg Asp Ala Ile Ala Glu Pro ValPro Pro Pro Ala Leu Ala Asp 1 5 10 15 Thr Pro Ala Ala Ala Met Glu GluLeu Arg Pro Ala Pro Pro Pro Gln 20 25 30 Pro Glu Pro Asp Pro Glu Cys CysPro Ala Ala Arg Gln Glu Cys Met 35 40 45 Leu Gly Glu Ser Ala Arg Lys SerMet Glu Ser Asp Pro Glu Asp Phe 50 55 60 Ser Asp Glu Thr Asn Thr Glu ThrLeu Tyr Gly Thr Ser Pro Pro Ser 65 70 75 80 Thr Pro Arg Gln Met Lys ArgLeu Ser Ala Lys His Gln Arg Asn Ser 85 90 95 Ala Gly Arg Pro Ala Ser ArgSer Asn Leu Lys Glu Lys Met Asn Thr 100 105 110 Pro Ser Gln Ser Pro HisLys Asp Leu Gly Lys Gly Val Glu Thr Val 115 120 125 Glu Glu Tyr Ser TyrLys Gln Glu Lys Lys Ile Arg Ala Thr Leu Arg 130 135 140 Thr Thr Glu ArgAsp His Lys Lys Asn Ala Gln Cys Ser Phe Met Leu 145 150 155 160 Asp SerVal Ala Gly Ser Leu Pro Lys Lys Ser Ile Pro Asp Val Asp 165 170 175 LeuAsn Lys Pro Tyr Leu Ser Leu Gly Cys Ser Asn Ala Lys Leu Pro 180 185 190Val Ser Met Pro Met Pro Ile Ala Arg Thr Ala Arg Gln Thr Ser Arg 195 200205 Thr Asp Cys Pro Ala Asp Arg Leu Lys Phe Phe Glu Thr Leu Arg Leu 210215 220 Leu Leu Lys Leu Thr Ser Val Ser Lys Lys Lys Asp Arg Glu Gln Arg225 230 235 240 Gly Gln Glu Asn Thr Ala Ala Phe Trp Phe Asn Arg Ser AsnGlu Leu 245 250 255 Ile Trp Leu Glu Leu Gln Ala Trp His Ala Gly Arg ThrIle Asn Asp 260 265 270 Gln Asp Leu Phe Leu Tyr Thr Ala Arg Gln Ala IlePro Asp Ile Ile 275 280 285 Asn Glu Ile Leu Thr Phe Lys Val Asn Tyr GlySer Ile Ala Phe Ser 290 295 300 Ser Asn Gly Ala Gly Phe Asn Gly Pro LeuVal Glu Gly Gln Cys Arg 305 310 315 320 Thr Pro Gln Glu Thr Asn Arg ValGly Cys Ser Ser Tyr His Glu His 325 330 335 Leu Gln Arg Gln Arg Val SerPhe Glu Gln Val Lys Arg Ile Met Glu 340 345 350 Leu Leu Glu Tyr Met GluAla Leu Tyr Pro Ser Leu Gln Ala Leu Gln 355 360 365 Lys Asp Tyr Glu ArgTyr Ala Ala Lys Asp Phe Glu Asp Arg Val Gln 370 375 380 Ala Leu Cys LeuTrp Leu Asn Ile Thr Lys Asp Leu Asn Gln Lys Leu 385 390 395 400 Arg IleMet Gly Thr Val Leu Gly Ile Lys Asn Leu Ser Asp Ile Gly 405 410 415 TrpPro Val Phe Glu Ile Pro Ser Pro Arg Pro Ser Lys Gly Tyr Glu 420 425 430Pro Glu Asp Glu Val Glu Asp Thr Glu Val Glu Leu Arg Glu Leu Glu 435 440445 Ser Gly Thr Glu Glu Ser Asp Glu Glu Pro Thr Pro Ser Pro Arg Val 450455 460 Pro Glu Leu Arg Leu Ser Thr Asp Thr Ile Leu Asp Ser Arg Ser Gln465 470 475 480 Gly Cys Val Ser Arg Lys Leu Glu Arg Leu Glu Ser Glu GluAsp Ser 485 490 495 Ile Gly Trp Gly Thr Ala Asp Cys Gly Pro Glu Ala SerArg His Cys 500 505 510 Leu Thr Ser Ile Tyr Arg Pro Phe Val Asp Lys AlaLeu Lys Gln Met 515 520 525 Gly Leu Arg Lys Leu Ile Leu Arg Leu His LysLeu Met Asn Gly Ser 530 535 540 Leu Gln Arg Ala Arg Val Ala Leu Val LysAsp Asp Arg Pro Val Glu 545 550 555 560 Phe Ser Asp Phe Pro Gly Pro MetTrp Gly Ser Asp Tyr Val Gln Leu 565 570 575 Ser Gly Thr Pro Pro Ser SerGlu Gln Lys Cys Ser Ala Val Ser Trp 580 585 590 Glu Glu Leu Arg Ala MetAsp Leu Pro Ser Phe Glu Pro Ala Phe Leu 595 600 605 Val Leu Cys Arg ValLeu Leu Asn Val Ile His Glu Cys Leu Lys Leu 610 615 620 Arg Leu Glu GlnArg Pro Ala Gly Glu Pro Ser Leu Leu Ser Ile Lys 625 630 635 640 Gln LeuVal Arg Glu Cys Lys Glu Val Leu Lys Gly Gly Leu Leu Met 645 650 655 LysGln Tyr Tyr Gln Phe Met Leu Gln Glu Val Leu Gly Gly Leu Glu 660 665 670Lys Thr Asp Cys Asn Met Asp Ala Phe Glu Glu Asp Leu Gln Lys Met 675 680685 Leu Met Val Tyr Phe Asp Tyr Met Arg Ser Trp Ile Gln Met Leu Gln 690695 700 Gln Leu Pro Gln Ala Ser His Ser Leu Lys Asn Leu Leu Glu Glu Glu705 710 715 720 Trp Asn Phe Thr Lys Glu Ile Thr His Tyr Ile Arg Gly GlyGlu Ala 725 730 735 Gln Ala Gly Lys Leu Phe Cys Asp Ile Ala Gly Met LeuLeu Lys Ser 740 745 750 Thr Gly Ser Phe Leu Glu Ser Gly Leu Gln Glu SerCys Ala Glu Leu 755 760 765 Trp Thr Ser Ala Asp Asp Asn Gly Ala Ala AspGlu Leu Arg Arg Ser 770 775 780 Val Ile Glu Ile Ser Arg Ala Leu Lys GluLeu Phe His Glu Ala Arg 785 790 795 800 Glu Arg Ala Ser Lys Ala Leu GlyPhe Ala Lys Met Leu Arg Lys Asp 805 810 815 Leu Glu Ile Ala Ala Glu PheVal Leu Ser Ala Ser Ala Arg Glu Leu 820 825 830 Leu Asp Ala Leu Lys AlaLys Gln Tyr Val Lys Val Gln Ile Pro Gly 835 840 845 Leu Glu Asn Leu HisVal Phe Val Pro Asp Ser Leu Ala Glu Glu Lys 850 855 860 Lys Ile Ile LeuGln Leu Leu Asn Ala Ala Thr Gly Lys Asp Cys Ser 865 870 875 880 Lys AspPro Asp Asp Val Phe Met Asp Ala Phe Leu Leu Leu Thr Lys 885 890 895 HisGly Asp Arg Ala Arg Asp Ser Glu Asp Gly Trp Gly Thr Trp Glu 900 905 910Ala Arg Ala Val Lys Ile Val Pro Gln Val Glu Thr Val Asp Thr Leu 915 920925 Arg Ser Met Gln Val Asp Asn Leu Leu Leu Val Val Met Glu Ser Ala 930935 940 His Leu Val Leu Gln Arg Lys Ala Phe Gln Gln Ser Ile Glu Gly Leu945 950 955 960 Met Thr Val Arg His Glu Gln Thr Ser Ser Gln Pro Ile IleAla Lys 965 970 975 Gly Leu Gln Gln Leu Lys Asn Asp Ala Leu Glu Leu CysAsn Arg Ile 980 985 990 Ser Asp Ala Ile Asp Arg Val Asp His Met Phe ThrLeu Glu Phe Asp 995 1000 1005 Ala Glu Val Glu Glu Ser Glu Ser Ala ThrLeu Gln Gln Tyr Tyr Arg 1010 1015 1020 Glu Ala Met Ile Gln Gly Tyr AsnPhe Gly Phe Glu Tyr His Lys Glu 1025 1030 1035 1040 Val Val Arg Leu MetSer Gly Glu Phe Arg Gln Lys Ile Gly Asp Lys 1045 1050 1055 Tyr Ile SerPhe Ala Gln Lys Trp Met Asn Tyr Val Leu Thr Lys Cys 1060 1065 1070 GluSer Gly Arg Gly Thr Arg Pro Arg Trp Ala Thr Gln Gly Phe Asp 1075 10801085 Phe Leu Gln Ala Ile Glu Pro Ala Phe Ile Ser Ala Leu Pro Glu Asp1090 1095 1100 Asp Phe Leu Ser Leu Gln Ala Leu Met Asn Glu Cys Ile GlyHis Val 1105 1110 1115 1120 Ile Gly Lys Pro His Ser Pro Val Thr Ala IleHis Arg Asn Ser Pro 1125 1130 1135 Arg Pro Val Lys Val Pro Arg Cys HisSer Asp Pro Pro Asn Pro His 1140 1145 1150 Leu Ile Ile Pro Thr Pro GluGly Phe Ser Thr Arg Ser Val Pro Ser 1155 1160 1165 Asp Ala Arg Thr HisGly Asn Ser Val Ala Ala Ala Ala Ala Val Arg 1170 1175 1180 Ala Ala AlaThr Thr Ala Ala Gly Arg Pro Gly Pro Gly Gly Gly Asp 1185 1190 1195 1200Ser Val Pro Ala Lys Pro Val Asn Thr Ala Pro Asp Thr Arg Gly Ser 12051210 1215 Ser Val Pro Glu Asn Asp Arg Leu Ala Ser Ile Ala Ala Glu LeuGln 1220 1225 1230 Phe Arg Ser Leu Ser Arg His Ser Ser Pro Thr Glu GluArg Asp Glu 1235 1240 1245 Pro Ala Tyr Pro Arg Ser Asp Ser Ser Gly SerThr Arg Arg Ser Trp 1250 1255 1260 Glu Leu Arg Thr Leu Ile Ser Gln ThrLys Asp Ser Ala Ser Lys Gln 1265 1270 1275 1280 Gly Pro Ile Glu Ala IleGln Lys Ser Val Arg Leu Phe Glu Glu Arg 1285 1290 1295 Arg Tyr Arg GluMet Arg Arg Lys Asn Ile Ile Gly Gln Val Cys Asp 1300 1305 1310 Thr ProLys Ser Tyr Asp Asn Val Met His Val Gly Leu Arg Lys Val 1315 1320 1325Thr Phe Lys Trp Gln Arg Gly Asn Lys Ile Gly Glu Gly Gln Tyr Gly 13301335 1340 Lys Val Tyr Thr Cys Ile Ser Val Asp Thr Gly Glu Leu Met AlaMet 1345 1350 1355 1360 Lys Glu Ile Arg Phe Gln Pro Asn Asp His Lys ThrIle Lys Glu Thr 1365 1370 1375 Ala Asp Glu Leu Lys Ile Phe Glu Gly IleLys His Pro Asn Leu Val 1380 1385 1390 Arg Tyr Phe Gly Val Glu Leu HisArg Glu Glu Met Tyr Ile Phe Met 1395 1400 1405 Glu Tyr Cys Asp Glu GlyThr Leu Glu Glu Val Ser Arg Leu Gly Leu 1410 1415 1420 Gln Glu His ValIle Arg Leu Tyr Thr Lys Gln Ile Thr Val Ala Ile 1425 1430 1435 1440 AsnVal Leu His Glu His Gly Ile Val His Arg Asp Ile Lys Gly Ala 1445 14501455 Asn Ile Phe Leu Thr Ser Ser Gly Leu Ile Lys Leu Gly Asp Phe Gly1460 1465 1470 Cys Ser Val Lys Leu Lys Asn Asn Ala Gln Thr Met Pro GlyGlu Val 1475 1480 1485 Asn Ser Thr Leu Gly Thr Ala Ala Tyr Met Ala ProGlu Val Ile Thr 1490 1495 1500 Arg Ala Lys Gly Glu Gly His Gly Arg AlaAla Asp Ile Trp Ser Leu 1505 1510 1515 1520 Gly Cys Val Val Ile Glu MetVal Thr Gly Lys Arg Pro Trp His Glu 1525 1530 1535 Tyr Glu His Asn PheGln Ile Met Tyr Lys Val Gly Met Gly His Lys 1540 1545 1550 Pro Pro IlePro Glu Arg Leu Ser Pro Glu Gly Lys Ala Phe Leu Ser 1555 1560 1565 HisCys Leu Glu Ser Asp Pro Lys Ile Arg Trp Thr Ala Ser Gln Leu 1570 15751580 Leu Asp His Ala Phe Val Lys Val Cys Thr Asp Glu Glu 1585 1590 159514 20 DNA Artificial Sequence synthetic construct 14 gaacaccatccagaagtttg 20 15 21 DNA Artificial Sequence synthetic construct 15cactttgtag acagggtcag c 21 16 25 DNA Artificial Sequence syntheticconstruct 16 tgggtcgcct ctgtcttata gacag 25 17 20 DNA ArtificialSequence synthetic construct 17 cacatcctgt gcttggtaac 20 18 21 DNAArtificial Sequence synthetic construct 18 aggacaagtg caggttagat g 21 1921 DNA synthetic construct 19 gctgtccata tctacagtgc t 21 20 21 DNAArtificial Sequence synthetic construct 20 cggcctggaa gcacgagtgg t 21 2121 DNA Artificial Sequence synthetic construct 21 ttcatccttg atgctgttttc 21 22 17 DNA Artificial Sequence synthetic construct 22 ggccagctcggtggcct 17 23 17 DNA Artificial Sequence synthetic construct 23tctggaatgt atcctgg 17 24 17 DNA Artificial Sequence synthetic construct24 agagaggaaa aagcggc 17 25 17 DNA Artificial Sequence syntheticconstruct 25 cagccagctc tcttccg 17 26 17 DNA Artificial Sequencesynthetic construct 26 ggaaaagtct tccgacc 17 27 21 DNA ArtificialSequence synthetic construct 27 ggccaaggag cttttggtag g 21 28 21 DNAArtificial Sequence synthetic construct 28 ggagctggtg gaggaccgaa g 21 2921 DNA Artificial Sequence synthetic construct 29 cccagaaccc tggccgaagct 21 30 21 DNA Artificial Sequence synthetic construct 30 agcacggtcccgcaggcagc c 21 31 21 DNA Artificial Sequence synthetic construct 31gtagtcgcca ccgccgcctc c 21 32 21 DNA Artificial Sequence syntheticconstruct 32 ctgacaagga attttcggca c 21 33 21 DNA Artificial Sequencesynthetic construct 33 accgccgcct ccgccatcgc c 21 34 21 DNA ArtificialSequence synthetic construct 34 cactgttcgc tggtctctgg g 21 35 21 DNAArtificial Sequence synthetic construct 35 agacaagcaa ggaggtgagt g 21 3621 DNA Artificial Sequence synthetic construct 36 gcctgacagc agccccttgcc 21 37 21 DNA Artificial Sequence synthetic construct 37 tccagttgctaaagaacttg c 21 38 21 DNA Artificial Sequence synthetic construct 38tggcagctgg cagcctgata g 21

I claim:
 1. An isolated nucleic acid molecule comprising a nucleotidesequence encoding a human MEKK protein, said MEKK protein having anamino acid sequence at least 98% identical to the amino acid sequence ofSEQ ID NO:2, wherein % identity is determined according to the ALIGNalgorithm using a PAM120 weight residue table, a gap length penalty of12 and a gap penalty of
 4. 2. An isolated nucleic acid molecule whichcomprises the nucleotide sequence of SEQ ID NO:1.
 3. The nucleic acidmolecule of claim 1, which encodes a MEKK protein having an amino acidsequence at least 99% identical to amino acid sequence of SEQ ID NO:2.4. The nucleic acid molecule of claim 1, which encodes a MEKK proteinhaving an amino acid sequence at least 99.5% identical to the amino acidsequence of SEQ ID NO:2.
 5. An isolated human MEKK protein comprising anamino acid sequence at least 98% identical to the amino acid sequence ofSEQ ID NO:2, wherein % identity is determined according to the ALIGNalgorithm using a PAM120 weight residue table, a gap length penalty of12 and a gap penalty of
 4. 6. An isolated protein comprising the aminoacid sequence of SEQ ID NO:2.
 7. The protein of claim 5, which comprisesan amino acid sequence at least 99% identical to the amino acid sequenceof SEQ ID NO:2.
 8. The protein of claim 5, which comprises an amino acidsequence at least 99.5% identical to the amino acid sequence of SEQ IDNO:2.
 9. An isolated nucleic acid molecule which encodes a MEKK proteinhaving the amino acid sequence of SEQ ID NO:2.
 10. An isolated nucleicacid molecule comprising a nucleotide sequence encoding a human MEKKprotein, said human MEKK protein having an amino acid sequence at least99.5% identical to the amino acid sequence of SEQ ID NO:4, wherein %identity is determined according to the ALIGN algorithm using a PAM120weight residue table, a gap length penalty of 12 and a gap penalty of 4.11. An isolated nucleic acid molecule which comprises the nucleotidesequence of SEQ ID NO:3.
 12. An isolated nucleic acid molecule whichencodes a MEKK protein having the amino acid sequence of SEQ ID NO:4.13. An isolated nucleic acid molecule comiprising a nucleotide sequenceencoding a human MEKK protein, said human MEKK protein having an aminoacid sequence at least 98% identical to the amino acid sequence of SEQID NO:6, wherein % identity is determined according to the ALIGNalgorithm using a PAM120 weight residue table, a gap length penalty of12 and a gap penalty of
 4. 14. An isolated nucleic acid molecule whichcomprises the nucleotide sequence of SEQ ID NO:5.
 15. The nucleic acidmolecule of claim 13, which encodes a MEKK protein having an amino acidsequence at least 99% identical to amino acid sequence of SEQ ID NO:6.16. The nucleic acid molecule of claim 13, which encodes a MEKK proteinhaving an amino acid sequence at least 99.5% identical to the amino acidsequence of SEQ ID NO:6.
 17. An isolated nucleic acid molecule whichencodes a MEKK protein having the amino acid sequence of SEQ ID NO:6.18. An isolated human MEKK protein comprising an amino acid sequence atleast 99.5% identical to the amino acid sequence of SEQ ID NO:4, wherein% identity is determined according to the ALIGN alogarithm using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of
 4. 19. An isolated protein comprising the amino acid sequenceof SEQ ID NO:4.
 20. An isolated human MEKK protein comprising an aminoacid sequence at least 98% identical to the amino acid sequence of SEQID NO:6, wherein % identity is determined according to the ALIGNalogarithm using a PAM120 weight residue table, a gap length penalty of12 and a gap penalty of
 4. 21. An isolated protein comprising the aminoacid sequence of SEQ ID NO:6.
 22. The protein of claim 20, whichcomprises an amino acid sequence at least 99% identical to the aminoacid sequence of SEQ ID NO:6.
 23. The protein of claim 20, whichcomprises an amino acid sequence at least 99.5% identical to the aminoacid sequence of SEQ ID NO:6.
 24. A vector comprising the nucleic acidmolecule of any one of claims 1-2, 3-4, 9-11 or 12-14.
 25. The vector ofclaim 5, which is an expression vector.
 26. A host cell containing thevector of claim
 25. 27. A method for producing a human MEKK proteincomprising culturing the host cell of claim 26 in a suitable mediumuntil a human MEKK protein is produced.
 28. The method of claim 27,further comprising isolating the human MEKK protein from the medium orthe host cell.
 29. A fusion protein comprising the MEKK protein of anyone of claims 5-6, 12-13, 18-19 or 20-23 operatively linked to apolypeptide other than MEKK.